RRM
2009 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract not available online - please check the printed booklet.
Abstract:
Structured RNA molecules form complex and compact folds composed of helices and hairpin, internal or junction \
References:
1. Leontis, N. B., Stombaugh, J., & Westhof, E. (2002). The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res, 30(16), 3497-3531.
2. Sarver, M., Zirbel, C. L., Stombaugh, J., Mokdad, A., & Leontis, N. B. (2008). FR3D: finding local and composite recurrent structural motifs in RNA 3D structures. J Math Biol, 56(1-2), 215-252.
3. Schuwirth, B. S., Borovinskaya, M. A., Hau, C. W., Zhang, W., Vila-Sanjurjo, A., Holton, J. M., et al. (2005). Structures of the bacterial ribosome at 3.5 A resolution. Science, 310(5749), 827-834.
4. Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., et al. (2000). Structure of the 30S ribosomal subunit. Nature, 407(6802), 327-339.
Keywords: RNA basepair, RNA basetriple, FR3D
Abstract:
The Arabidopsis ortholog of the 30 kDa subunit of the cleavage and polyadenylation factor (AtCPSF30) is an RNA binding endonuclease, and the endonuclease activity is inhibited by reducing agents. Here, we report the presence of a disulfide linkage in the endonuclease motif. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to compare the reduced and non-reduced protein. This analysis reveals presence of a disulfide bond within the third predicted CCCH -zinc finger, the motif that catalyzes the endonuclease activity of AtCPSF30. This raises the possibility that redox regulation of AtCPSF30 may occur through the disulfide linkage.
References:
1. Delaney, K.J., Xu, R., Zhang, J., Li, Q.Q., Yun, K.Y., Falcone, D.L. and Hunt, A.G. (2006). Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiology 140, 1507-1521.
2. Addepalli, B. and Hunt, A.G. (2007). A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor. Nucleic Acids Research 35, 4453-4463.
3. Zhang, J., Addepalli, B., Yun, K.Y., Hunt, A.G., Xu, R., Rao, S., Li, Q.Q. and Falcone, D.L. (2008). A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana. PLoS ONE 3
4. Addepalli, B. and Hunt, A.G. (2008). Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit. Archives of Biochemistry and Biophysics 473, 88-95.
Keywords: CPSF30, Endonuclease, Disulfide linkage
Abstract:
A large number of miRNA genes are encoded in U2-dependent spliceosomal introns of mammalian genes. Current evidence suggests that the processing of intronic miRNA does not affect the splicing. Since, intronic miRNAs are involved in essential cellular processes, we surmised if splice sites of miRNA coding introns are flexible to support productive primary and precursor miRNA processing. Using in vivo and in vitro methods, we examined the splicing of á-myosin heavy chain intron 27 and processing of miR-208, a resident of intron 27. The 5' splice site mutations of intron 27 do not affect primary miRNA processing. Complete elimination of 5' splice site activated a cryptic splicing. Interestingly, conversion of the U2-type 5' splice site to consensus U12-type 5' splice site did not affect in vivo splicing of the intron, suggesting potential conversion of the intron to U12-type, albeit without a consensus U12-type branch site sequence. To test if this intron is spliced by minor spliceosome, we mutated CC5/6GG of the 5' splice site, which is essential for U12-dependent splicing, as the sequence is recognized by U11 and U6atac snRNAs sequentially. Surprisingly, these mutations neither completely abolished the splicing nor activated any cryptic splicing. Co-expression of appropriate U6atac and U11 suppressors results support the notion that the intron might be spliced by a combination of major and minor spliceosomal snRNAs. All known miRNAs are harbored in U2-dependent introns only; we wanted to check if miRNA can process from a U12-dependent spliceosomal intron. To test this we cloned miR 208 in a U12-depedent intron and assayed it for in vivo splicing. To our surprise, the intron spliced in in vivo system and miR208 got processed from the intron as detected by in vitro splicing assay. We also mutated the 5’ splice site of intron 27 at positions 3’ to 7’ individually to check the effect of these mutations on splicing of U2-dependent intron containing miRNA. Experiments are being pursued to determine if the plasticity of splice sites is an evolutionary derived feature of introns which code for miRNA genes.
Keywords: Splicing, MicroRNA, miR processing
Abstract not available online - please check the printed booklet.
Abstract:
Fluorescent intercalator displacement (FID) is becoming an important tool for identifying new nucleic acid binding ligands. Its success is based on the fact that it can be fashioned into a versatile high-throughput assay that can be used for assessing the relative binding affinities of compounds to nucleic acids in a simple and efficient manner, and requiring no significant expertise. FID is becoming an increasingly attractive method because it is a tag-less approach; neither the RNA nor the small molecule under investigation has to be modified.
In this study, an FID method for screening RNA-binding ligands was established, using TO-PRO as the fluorescent intercalator. Electrospray ionization mass spectrometry (ESI-MS) was used to investigate its mechanism, and the assay was then successfully applied to screening a variety of RNA-binding ligands.
Our assay was able to differentiate ligands that bind to a variety of RNA constructs used in this study (A-site, TAR, H31 and H69) with moderate selectivity. Furthermore, the FID results were compared to those obtained using ESI-MS. Our results provide molecular evidence that correlates the reduction in fluorescence observed in the FID assay with the displacement of a dye molecule from RNA.
Keywords: Fluorescent intercalator displacement, Electrospray ionization mass spectrometry, RNA
Abstract not available online - please check the printed booklet.
Abstract:
Two families of RNA binding proteins that are involved in splicing regulation are the CUG-BP and ETR-3 like factors (CELF) and the Muscleblind-like proteins (MBNL). The CELF protein family consists of six highly conserved family members and are known to bind to UG-rich RNA elements. The MBNL family has three family members and bind to the YGCU(U/G)Y motif (Y, pyrimidine) with a preference for stem-loop RNA secondary structures. Although these proteins have distinct splicing targets, MBNL and CELF proteins act as antagonists in six pre-mRNA targets. We have identified the neurofibromatosis type I (NF1) pre-mRNA as a novel target of CELF protein-mediated splicing regulation. The NF1 mRNA encodes a 2,800 amino acid protein called neurofibromin. Exon 23a, which is skipped in neuronal tissues and included in other tissue types, is especially interesting, because it falls within the GAP-related domain of the NF1 protein. The type II isoform, which contains exon 23a, is associated with low GAP activity while the type I isoform, in which exon 23a is skipped, is associated with high GAP activity. We show that over-expression of CELF proteins in HeLa cells promoted skipping of exon 23a in both a NF1 splicing reporter and the endogenous NF1. SiRNA knockdown of endogenous ETR-3 knockdown promoted inclusion of NF1 exon 23a. Over-expression of a dominant-negative CELF protein promoted inclusion of exon 23a, in CA77 cells, providing further evidence that CELF proteins function as negative regulators of NF1 exon 23a. UV-crosslinking immunoprecipitation assays have confirmed that CUG-BP1 and ETR-3 bind, strongly, to the NF1 pre-mRNA. In vitro splicing and binding analyses show that CELF proteins block splicing of NF1 exon 23a. We have identified two MBNL binding motifs in the intronic sequence upstream to NF1 exon 23a. The RNA sequence within this region is predicted to form a stem-loop structure. Over-expression of MBNL1 promoted NF1 exon 23a inclusion in CA77 cells, suggesting that they are positive regulators. Future work will focus on the regulation of this event by MBNL and how MBNL and CELF proteins function as antagonists.
Keywords: Alternative Splicing, Neurofibromatosis Type I, CELF and MBNL
Abstract:
Most eukaryotic nuclear-encoded genes are interrupted by introns, which are needed to be removed from pre-mRNAs by splicing to generate functional mRNAs. In metazoan, two types of introns are spliced by two distinct spliceosomes. The major type of introns or U2-dependent introns are spliced by U2-dependent spliceosomal small nuclear (sn) RNAs including U1, U2, U4, U5 and U6. On the other hand, minor or U12-dependent introns are spliced by a distinct set of snRNAs containing U11, U12, U4atac and U6atac snRNAs. U5 appears to be a common snRNA in both systems. Recent work shows that 3’ RNA element of U6atac snRNA is sufficient to guide U6 snRNA to minor spliceosome and activates the splicing of a U12-dependent intron. This data lead us to believe that splicing activity that targets U6atac to minor spliceosome is modulated by a RNA binding protein specific to 3’ stem-loop element of RNA element. Of various novel proteins of U12-dependent spliceosome, protein 65K 3’RRM contains a well–characterized domain called 3’ -RNA recognition motif and our preliminary data indicate that P65 3’ RRM protein has potential to bind U6atac 3’ stem-loop element. To determine 65K-C-RRM protein-RNA binding site on U6atac 3’ stem-loop, we are using Electrophoretic Mobility Shift Assay (EMSA) and a series of deletion mutants of U6atac RNAs. More investigations need to be done to determine dual binding specificity of 65kd RNA binding protein in the minor spliceosome.
Keywords: P65 3 RRM protein, U12-dependent spliceosome, U6atac 3 stem-loop element
Abstract not available online - please check the printed booklet.
Abstract:
The 77 kDa subunit of the heterotrimeric complex known as the cleavage stimulation factor (CstF-77) plays an integral role in messenger RNA 3’end processing. Previous studies have revealed the C-terminus of the Arabidopsis ortholog of CstF-77 (AtCstF-77) exhibits protein-protein interactions with the N-terminus of AtCstF-64 and an Arabidopsis Fip1 (Factor Interacting with Poly-A polymerase) ortholog (1,2). In addition, it has been demonstrated through yeast two-hybrid analyses that AtCstF-77 interacts with the Arabidopsis ortholog of the 30 kD subunit of the Cleavage and Polyadenylation Specificity Factor (AtCPSF-30) (3). To further understand this latter interaction, the crystal structure of mammalian CstF-77 was used to appropriately divide AtCstF-77 into three regions: the N-terminus, Middle, and C-terminus (4). In vitro pull-down assays were carried out with each region to determine the region(s) binding AtCPSF-30. These experiments demonstrated that that the C-terminus of At-CstF-77 interacts with AtCPSF-30, confirming previous studies. As AtCPSF-30 is an RNA-binding protein, the effects of AtCstF-77 on RNA binding by AtCPSF-30 were assayed. Remarkably, in the course of these studies, it was found that the C-terminus of AtCstF-77 by itself possesses RNA-binding activity. This activity seemed non-specific in terms of RNA sequence requirements since RNAs lacking the plant polyadenylation signal sub-elements (near-upstream, far-upstream, and cleavage elements) were all equally effective in binding to AtCstF-77. These studies therefore reveal AtCstF-77 to be an RNA binding protein. This adds yet another RNA-binding activity to the plant polyadenylation complex, and raises interesting questions as to the means by which RNAs are recognized and handled in the course of mRNA 3’ end formation in plants.
References:
1. Yao Y, Song L, Katz Y, Galili G: Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3\' mRNA cleavage and polyadenylation. J Exp Bot 2002, 53:2277-2278.
2. Forbes KP, Addepalli B, Hunt AG: An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits. J Biol Chem 2006, 281:176-186.
3. Hunt AG, Xu R, Adepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhyay A, Dampanaboina L, Marion A, Von Lanken C, Li QQ: Arabidopsis mRNA polyadenylation machinery: a comprehensis analysis of protein-protein interactions and gene expression profiling. BMC Genomics 2008, 9:220.
4. Bai Y. Auperin TC, Chou CY, Chang GG, Manley J, Tong L: Crystal Structure of Murine CstF-77: Dimeric Association and Implications for Polyadenylation of mRNA Precursors. Molecular Cell 2007, 25: 863-875.
Keywords: polyadenylation, RNA-binding, CstF-77, and CPSF-30
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Truncated mRNAs missing a stop codon cause synthesis of defective proteins and trap
ribosomes at the end of their incomplete message. Stalled ribosomes can cause 2 major
problems: the defective unfinished protein can be toxic for the cell, and the pool of
ribosomes available for translation is depleted. In bacteria, both problems can be solved
by a rescue process called trans-translation, which employs transfer-messenger RNA
(tmRNA) and its cofactor the small protein B (SmpB). Previous studies have shown the
importance of tmRNA during gene expression and for virulence in many pathogenic
bacteria, making it an attractive target for antimicrobial drug discovery. However, key
aspects of this mechanism are still not fully understood.
Here, we have used fluorescence anisotropy and fluorescence resonance energy transfer
to characterize the strucuture of the tmRNA-SmpB complex. Our results show that SmpB
binds the TLD domain of tmRNA tightly and specifically. However, we did not observe
any conformational change in the global structure of the tmRNA in complex with SmpB.
Furthermore, the distance between the acceptor stem and the irregular helix of the
tmRNA-SmpB complex is ~ 5 Å shorter than in regular tRNA, in contradiction with a
previously proposed hypothesis based on a recent crystal structure. This result suggests
that tmRNA-SmpB complex maybe compact enough to fit freely into the A site of the
ribosome.
Keywords: tmRNA, SmpB, structural Characterization
Abstract:
We have shown that the release of ribosomal protein L13a from 60S ribosome and its subsequent phosphorylation is a key step in the formation of GAIT complex in transcript specific translational silencing. In order to gain insight into the mechanism of release, it is necessary to determine the domain of L13a responsible for binding to 60S ribosome. Using the ribosome incorporation assay of the ectopically expressed tagged L13a we have identified ribosome-incorporation defective mutants of L13a. We have performed structural homology modeling of human L13a based on the crystal structure of prokaryotic L13 (the homolog of mammalian L13a), which identified a loop in L13a, harboring Arg at position 68 at the tip of the loop. in silico analysis of L13a has predicted the amino acids most likely to contact RNA in solved complex structure from protein data bank. This analysis also identified several candidate amino acid residues and motif that potentially bind to rRNA. In consistence with the above two models, we have experimentally verified Arg 68 and triple mutant Arg-Lys-Arg, 59-60-61 as ribosome incorporation defective L13a. We then tested the role of other predicted amino acid residues, which showed no incorporation defect. We will confirm the incorporation defective mutants by testing the in vitro direct binding between purified 60S ribosome and recombinant His tagged L13a using sucrose gradient analysis. In consistence with the ribosome incorporation assay our recent result using immunoprecipitation coupled with RT-PCR shows direct binding between wild-type L13a, however Arg 68 mutant failed to bind. Imaging studies show no defect in the nuclear translocation step of the mutant. Interestingly no difference was observed in the IFN-γ mediated delayed phosphorylation between the wild-type or incorporation defective mutant.
Keywords: L13a, Incorporation Defect, Direct Binding
Abstract:
The long term goal of this research is to understand the significance of posttranscriptional modification in ribosomal RNA (rRNA) and posttranslational modification in ribosomal proteins (r-proteins) within ribosome assembly defects. In particular, mass spectrometry-based approaches will be used to characterize and identify the components of a transient ribonucleoprotein complex that is formed when bacterial cells are treated with the antibiotic erythromycin.
Binding of erythromycin to the ribosome can both inhibit protein translation and the assembly of the ribosome. Specifically, it interferes with the assembly of the 50S subunit leading to a stalled intermediate which is a target of degradation by cellular RNases. The use of the mutant SK5665 strain of E. coli which has a temperature-sensitive RNase E phenotype, allows for the isolation of the improperly folded assembly intermediate. Initial MALDI-MS data of the r-proteins isolated from the intermediate showed the absence of several proteins and was confirmed by SDS PAGE electrophoresis. Optimal growth conditions for growing the mutant SK5665 strain as well as the purification of the intermediate by sucrose density gradient ultracentrifugation will be presented. Further work is focused on determining whether the ribosomal proteins present in the improperly folded assembly intermediate differ from those found in properly assembled 50S subunits, both in identity and modification status.
Keywords: ribosome assembly intermediates, ribosomal proteins, mass spectrometry
Abstract not available online - please check the printed booklet.
Abstract:
Upon HIV-1 budding from the host cell membrane during the late phase of the viral lifecycle, the proteolytic cleavage of the Gag polyprotein leads to the formation of the characteristic conical core formed by the viral capsid (CA) protein in the mature virus. In the cytoplasm prior to viral assembly, CA interacts with several host cell factors, including human lysyl-tRNA synthetase (LysRS). Binding to LysRS facilitates selection and packaging of host cell tRNALys,3, which is the primer for reverse transcription. The numerous functions of CA in the viral lifecycle make it an important antiviral target. Small-molecule inhibitors against HIV-1 CA, such as a recently identified peptide-based CA assembly inhibitor (CAI), have potential clinical applications. In this work, a robust method has been employed for the high-throughput synthesis, screening and identification of cyclic peptidyl ligands against HIV-1 CA. Support-bound cyclic peptide libraries containing randomized amino acid sequences and different ring sizes were synthesized and screened for binding to Texas Red-labeled full-length CA and a monomeric form of the CA C-terminal domain (WM-CA CTD). Selective inhibitors identified from the library showed strong specific binding (Kd ~ 500 nM range) to both CA and WM-CA CTD in vitro. Two of the selected peptides also inhibited LysRS and CA interaction in vitro with an IC50 value of ~ 1 µM. To demonstrate the in vivo efficacy, we have designed cell permeable peptides and these studies are currently underway. Ongoing work is also aimed at studying the cyclic peptide binding site on CA by NMR. As an additional approach to discover small molecule inhibitors, we have also employed SELEX to select for high-affinity RNA aptamers that bind to HIV-1 CA. Briefly, a 40-nt randomized RNA with 20-nt flanking primer binding site regions is being used to select for RNAs that bind to His6-CA immobilized on a Ni-NTA resin. Results of preliminary rounds of selection will be presented.
Keywords: CA, LysRS
Abstract:
Ribosomes are molecular machines essential to the process of life in all organisms because they are the instruments of the cell that perform translation of genetic information into proteins. Ribosomal RNA (rRNA) is particularly important, because it serves as the functional unit of ribosomes. The part of the ribosome of focus in our experiments is the decoding region – the area where the ribosome binds to transfer RNA (tRNA) – within the small ribosomal subunit (30S). In the decoding region, there are several modified bases. Although it is already known that these modifications are associated with antibiotic resistance and sensitivity, the mechanism and the function of modified nucleotides remain unclear. To understand the effects of these modified nucleotides, a new methodology to incorporate desired nucleosides into a specific site without inhibiting ribosomal activity is required. To date, several ways to engineer RNAs have been reported, but those methods are not optimized for ribosome engineering, and they are also cost ineffective. To develop a cost effective and powerful way for RNA engineering, we are focusing on the DNA enzyme (DNAzyme) which can cleave RNA at a specific site. We used two major types of DNAzyme, the 8-17 DNAzyme and the 10-23 DNAzyme, to test their enzyme activity on rRNA. The 8-17 DNAzymes target GG dinucleotides at positions 1904 of 23S rRNA and 1421 of 16S rRNA, and the 10-23 DNAzymes target GU and AU dinucleotides at positions 1392, 1413 of 16S rRNA, respectively. These DNAzymes show cleavage activities only after incubation at room temperature in the presence of divalent metal ions. We tested the catalytic effect of several divalent metal ions on RNA cleavage activity and concluded that Mg2+ is the best divalent metal ion for efficient cleavage of rRNA by the DNAzyme. Interestingly, we also observed significant DNAzyme reactivity without annealing DNAzymes to rRNA in advance. In conclusion, the DNAzyme has thus far proven effective in site-specifically cleaving the 16S rRNA. The next steps would be to purify the products of the DNAzyme reactions and continue with ligation of rRNA segments through the use of T4 DNA ligase.
Keywords: DNAzyme, RNA, ribosome
Abstract:
The hepatitis delta virus (HDV) replication cycle requires ribozyme mediated RNA cleavage. The cleavage mechanism is noteworthy for the involvement of an active site cytosine residue, C76, that acts as a general acid. The reaction pKa for the general acid C76 residue is significantly shifted by at least 2 pH units from the intrinsic pKa of cytosine. A putative network of hydrogen bonding interactions may alter the intrinsic pKa of C76 and allow it to act as a catalyst. We seek to determine the influence of the putative active site network that promotes the catalytic capability of the C76 residue. Here we replace two non-bridging oxygen atoms in an internucleotide phosphate within the network with sulfur atoms. Substitution of the oxygen atoms results in the RP and SP diastereomeric phosphorothioate RNAs which are separated by ion-exchange HPLC. These RNAs are each ligated to a transcribed RNA using T4 RNA ligase to provide two full-length single atom mutant ribozymes. The cleavage rate of each single atom mutant ribozyme will give insight into the influence of the pro-RP and pro-SP oxygen atoms in the active-site network on the catalytic cytosine residue.
Keywords: HDV ribozyme, chemo-genetic analysis
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Guanosine tetraphosphate (ppGpp) is one of the major regulators of stress response in bacteria. Numerous in vivo studies demonstrated large ppGpp effects on transcription, however, ppGpp alone displayed only minor effects in a purified in vitro system. To explain this discrepancy, the dnaK suppressor protein (DksA), was suggested to potentiate ppGpp effect in the cell. Indeed, DksA was shown to stimulate ppGpp activity in vitro. In this work, we performed a kinetic analysis of ppGpp and DksA effect at λPR promoter that provided several new insights:
A.ppGpp alone has a low affinity to the E. coli RNA polymerase (RNAP);
B.DksA inhibits transcription at the λPR promoter;
C.ppGpp increases DksA affinity to RNAP by more than 30-fold.
This analysis, together with the observations that, in contrast to ppGpp
whose levels rise dramatically during starvation, DksA is constitutively
expressed in the cell, led us to the following model:
Upon stress, ppGpp levels increase in the cell and consequently the
affinity of DksA to RNAP which leads to pleiotropic effects on
transcription. Interestingly different substitutions in DksA that increase
its affinity to RNAP may in part bypass the requirement for ppGpp.
References:
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Laptenko O. et al., EMBO J. (2006),25(10):2131-41.
Keywords: DksA, ppGpp, RNAP
Abstract:
CPSF30 in Medicago.
Bobby Gaffney
CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana. In this study the gene for CPSF30 has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana and both medicago species. This area of the gene was cloned out and 5’ and 3’ RACE were used to get each full length gene. The Medicago genes were cloned into a protein expression vector. The vector was used to translate each gene into its corresponding protein and each protein was purified and dialyzed. CPSF30 in A. thaliana binds calmodulin and it was hypothesized that CPSF30 from both the Medicago species would also bind calmodulin based on the conservation of the amino acids between the model species and the Medicago species. This study shows that CPSF30 from both Medicago species does bind calmodulin. CPSF30 from A. thaliana also has been shown to possess three CCCH type zinc fingers, with the first implicated in RNA binding and the third in RNA cleavage. Amino acid alignments showed the zinc finger regions were conserved and it was hypothesized that CPSF30 from both Medicago species would bind RNA and cleave RNA. Electromobility shift assays were used to determine that RNA binding did occur. RNA nuclease activity in Medicago is still being tested. CPSF30 knockout studies in A. thaliana have yielded some consistent phenotypes that are still being characterized and explored. One such characteristic is an elevated resistance to oxidative stress. CPSF30 knockouts are have been produced in Medicago sativa. The plants are being evaluated to determine what effects the absence of CPSF30 from M. sativa might have.
Keywords: Medicago, RNA Processing
Abstract not available online - please check the printed booklet.
Abstract:
In this study we provide evidence that Dcp2p, the decapping enzyme which destroys mRNAs in the cytoplasm, is involved in the destruction of noncoding RNAs (ncRNAs). We have preliminary evidence suggesting that abrogation of decapping stabilizes ncRNAs ultimately resulting in transcriptional downregulation.
In the cytoplasm, mRNA turnover is initiated by the loss of the poly(A) tail, followed by decapping, and subsequent 5’-3’ exoribonuclease digestion. Blocking mRNA decapping by mutation results in dramatic stabilization of the mRNA but no apparent increase in total mRNA levels at steady-state relative to wild-type cells. We account for this discrepancy by showing that loss of decapping enzyme function results in a dramatic inhibition of nuclear mRNA transcription at the well-studied GAL loci. First, in induction assays, decapping enzyme mutants accumulate significantly less message than a wild-type cell (WT). Additionally, appropriate transcription requires the catalytic activity of the decapping enzyme. Point mutations in the catalytic domain of DCP2, dcp2-4, result in induction defects similar to the gene deletion. Second, blocking cytoplasmic decapping or decay by other extragenic mutations (i.e. mutations in decapping activators or the cytoplasmic exonuclease) does not result in aberrant transcription. Third, ncRNAs specifically at the GAL loci, which have been implicated in transcriptional regulation, show increased levels in decapping enzyme mutants. Together these data are suggestive of a novel, possibly nuclear function for the decapping enzyme in regulating mRNA transcription through the destruction of ncRNAs.
Keywords: Decapping enzyme, mRNA decay, transcription
Abstract:
Despite a large body of information provided by X-ray analysis of prokaryotic ribosomes, the role of many prokaryotic ribosomal proteins remains rather obscure. Even less is known about the functions of eukaryotic ribosomal proteins. Many eukaryotic proteins have evolved additional segments, the function(s) of which are not quite clear, but may be related to the differences in translation machineries between different kingdoms. Very little is also known about the position of initiation factors on the surface of the 40S subunit and in particular the role of ribosomal proteins in recruiting these factors. Ribosomal protein (rp) S5 belongs to a family of conserved ribosomal proteins that includes bacterial rpS7. The protein forms part of the exit (E) site on the 30S/40S ribosomes and contributes to the formation of the mRNA exit channel. rpS5/7 proteins possess a conserved central/C-terminal region and variable N-terminal ends. Many eukaryotes and in particular fungi have evolved a longer rpS5 protein with substantial N-terminal extension (>50 aa). To investigate the function of the yeast rpS5 and in particular the role of its N-term region, we obtained and characterized yeast strains in which the wt yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. All mutant yeast strains were viable and displayed only moderately reduced growth rates, with the exception of the strain lacking 46 N-terminal amino acids, which had a doubling time of about 3 hours. Biochemical analysis of the mutant yeast strains suggests that the N-terminal part of the eukaryotic and in particular yeast rpS5 may impact the ability of 40S subunits to function properly in translation and affects the efficiency and accuracy of initiation process, in particular, increasing the affinity of eIF2 to mutant 40S ribosomes. We hypothesize that N-terminal extension of eukaryotic ribosomal protein S5 has evolved to control the interaction of eukaryotic initiation factor 2 (and perhaps some other factors) with the 40S ribosome.
Keywords: Ribosomal protein S5, translation, eIF2
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The increased prevalence of drug-resistant bacteria is a global human health problem. RNA-protein (RNP) complexes, which play a vital role in cellular processes, have merited scrutiny as candidate targets for novel antibacterials. RNase P, a catalytic RNP, is primarily responsible for the Mg2+-dependent removal of the 5’ leader sequence in all precursor tRNAs. Despite its essential and conserved function in tRNA biogenesis, the subunit composition of the RNase P holoenzyme varies in the three domains of life. All holoenzymes comprise an essential RNase P RNA (RPR) and a variable number of RNase P Protein (RPP) subunits: one, four, and nine in Bacteria, Archaea, and Eukarya, respectively. We have exploited the striking differences between the structure of RNase P in pathogenic bacteria and their eukaryotic hosts for designing new antibacterials. In this study, we hypothesized that certain synthetic arginine-rich, â-hairpin peptides, designed to disrupt the assembly of viral protein-RNA complexes, would also serve as structural mimics of a highly conserved helix in bacterial RPPs and disrupt bacterial RNase P assembly. Indeed, some of these cyclic peptides exhibited sub-micromolar IC50 values when tested in vitro for their ability to inhibit the ptRNA processing activity of recombinant Escherichia coli RNase P. Moreover, as the RPR and RPP are ~99% identical between the corresponding antibiotic-resistant and -sensitive variants of different pathogenic bacteria, we predicted and confirmed (using Salmonella typhi as a test case) that the MIC values (determined to be ~ 1 mM) for our most potent inhibitor are indistinguishable between resistant and sensitive strains. This active peptide also had no effect on growth of HeLa cells. Ongoing directions include determining the inhibitor’s mechanism of action and proving that cessation of bacterial growth in culture stems from decreased RNase P activity in vivo.
Keywords: RNase P
Abstract:
Human immunodeficiency virus (HIV) is a retrovirus that relies on nucleic acid (NA) chaperone proteins, which function to remodel NAs at critical steps throughout the viral lifecycle. Upon entering a host cell, HIV reverse transcribes its RNA genome into DNA, which is integrated into the host DNA. Following the production of new viral RNAs and proteins, the assembly of new virus particles takes place at the plasma membrane. The polyprotein Gag, which consists of the matrix, capsid, nucleocapsid (NC), and p6 domains, directs virus assembly and packages the viral RNA genome into new virions. Gag is a chaperone protein that is also responsible for annealing of a host cell tRNALys primer to the HIV genome, the first step of reverse transcription. After virus budding, Gag is proteolyzed, and freestanding NC assumes the role as the chaperone for the remaining steps of reverse transcription. Although both Gag and NC promote tRNALys annealing to the genome in vitro, we have found that extension of the primer by reverse transcriptase is inhibited by Gag. Interestingly, in vivo studies have found that virus variants with specific mutations in the NC domain of Gag undergo premature reverse transcription, which causes the packaging of DNA and abolishes infectivity. Thus, we hypothesize that Gag’s role in inhibiting reverse transcription depends upon critical structures within the NC domain of Gag, which remodel the primer/genome complex in such as way as to prevent premature reverse transcription. In this work, we begin to test this hypothesis by preparing Gag variants with mutations in the NC domain and testing their capability to aggregate and bind NA, destabilize NA secondary structure, and facilitate tRNA annealing and reverse transcription in vitro. We show that these mutants bind to NAs with reduced affinity relative to wild-type (WT) Gag. However, they aggregate and anneal NA as efficiently as WT Gag. Surprisingly, these mutants are better than WT Gag at destabilizing NA secondary structure. Studies to examine the effect of NC domain mutants on Gag’s inhibition of reverse transcription are underway.
Keywords: HIV, reverse transcription
Abstract:
Translocation of biopolymers such as RNA or DNA is an important theme in living systems. The connector channel of the phi 29 DNA-packaging motor is an intriguing element in this category. We demonstrated that the lipid-embedded connector channel has the ability to translocate double stranded and single stranded DNA or single stranded RNA under an applied volatge (Nature Nanotechnology, in press). Herein, we developed an automated system using MATLAB for detailed analysis of nucleic acids translocating through the protein nanopore using a set of parameters. Individual pore-blockade events are first distinguished from noise events with high confidence. The chracateristic depths and duration of the current blockade events are then determined to distinguish varying lengths and conformational structure of RNA or DNA. The automated single molecule RNA/DNA analysis has the potential for future high throughput analysis of various bioplomers as well as DNA sequencing applications.
Keywords: Bacteriophage phi29, Nano-Channel, RNA translocation
Abstract:
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common genetic disorders, affecting 1 in 500-1000 people. The disease is characterized by cyst formation on the kidneys that can lead to renal failure. The phenotypic severity of the disease varies among patients and the source of this variation is not well understood. ADPKD is caused by mutations in either the PKD1 or PKD2 gene. Our lab is characterizing a disease-associated C to T point mutation in the polypyrimidine tract of intron 45 of PKD1. Intron 45 is a mirtron—an intron that gives rise to a microRNA (miR-1225) in a splicing-dependent manner. The mutation in intron 45 may disrupt intron splicing and/or microRNA processing and targeting. Mir-1225 is predicted to target genes involved in several diseases including idiopathic kidney stones, Dent’s disease and Prader-Willi syndrome. Alterations in mir-1225 expression or activity could be a determinant of phenotypic severity in ADPKD. We find that the point mutation in intron 45 of PKD1/mir-1225 partially inhibits splicing and also leads to a reduction in mir-1225 abundance. We are currently investigating the relationship between splicing and mir-1225 expression and testing whether mir-1225 expression is altered in ADPKD patients. Understanding the consequences of this point mutation can help to elucidate the disease mechanism and potentially explain some of the variability in phenotypic severity. Furthermore, our characterization could help guide therapeutic development for disease treatment.
Keywords: MicroRNA, Polycystic Kidney Disease, splicing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In eukaryotes, tRNA is transcribed in the nucleus and functions in the cytoplasm. In the yeast Saccharomyces cerevisiae pre-tRNA splicing occurs on cytoplasmic surface of mitochondria and tRNA subcellular movement is not strictly unidirectional from the nucleus to the cytoplasm. However, much remains to be learned about tRNA nuclear-cytoplasmic dynamics. We are using genome-wide analyses and in vivo screening to identify novel gene products involved in tRNA processing and subcellular movement. We are employing Northern analysis to investigate the processing patterns of pre-tRNA from the yeast genome-wide deletion collection, screening for candidates that reproducibly evidence higher molar yield of intron-containing tRNAs relative to mature tRNAs. Such mutants are expected to either have defects in the pre-tRNA processing machinery and/or pre-tRNA subcellular distributions. So far, we identified mutations of mitochondrial and ribosomal protein-encoding genes. In a second approach we are investigating the β-importin family member, Exportin-5 (yeast Msn5), that has been implicated in tRNA subcellular dynamics. Exportin-5/Msn5 functions in nuclear export of micro-RNAs in metazoans and in nuclear export of particular phosphorylated nucleus/cytoplasmic shuttling proteins in yeast. Our data show that Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing tRNA, supporting the model that Msn5 functions solely in the re-export to the cytoplasm. To study the role of Msn5 in tRNA nuclear re-export, we are attempting to identify its binding partner in vivo. One possible binding partner is Tef1/2. TEF1 and TEF2 encode identical eukaryotic translation elongation factor 1 alpha. Tef1/2 bind aminoacyl-tRNAs and function in delivering aa-tRNA to ribosomes. Our studies show that in wild-type strains, Tef1-GFP is excluded from the nucleus, while in msn5Δ cells Tef1-GFP is localized in both cytoplasm and nucleus. This result suggests that Msn5 is responsible for exporting Tef1 from the nucleus and may be involved in the tRNA re-export process. To further investigate the interaction of Tef1 and Msn5, I will determine whether a Msn5•Tef1/2•tRNA•Ran-GTP complex assembles for tRNA re-export.
References:
1. Shaleen, H.H. and Hopper, A.K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11290-11295
2. Takano, A., Endo, T., Yoshihisa T. (2005) Science 309, 140-142
3. Yoshihisa, T., Yunoki-Esaki, K., Ohshima, C., Tanaka, N., Endo, T. (2003) Mol. Biol. Cell 14, 3266-3279
Keywords: tRNA processing
Abstract not available online - please check the printed booklet.
Abstract:
Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by mutations in the SMN1 gene, leading to motor neuron death and progressive muscle weakness and atrophy. SMN2 is nearly identical to SMN1 and both code for SMN protein. However, most of the mRNA produced from SMN2 lacks exon 7 and codes for a truncated, unstable protein product. SMN protein is an essential part of a complex that assembles Sm proteins onto spliceosomal snRNAs during the production of mature snRNPs that are required for splicing. We hypothesize that the loss of SMN1 in SMA leads to a reduction in snRNPs that consequently impacts SMN2 exon 7 splicing. We observe that high SMN protein levels correlate with greater SMN2 exon 7 splicing in mice and in induced pluripotent stem cells derived from a normal individual compared to cells from an SMA patient with lower SMN protein levels. These results suggest a link between SMN protein levels and exon 7 splicing, possibly via modulation of snRNP abundance. To test this idea, we inactivated individual snRNPs in vitro. We observed differential effects on exon 7 inclusion depending on which snRNP was targeted, suggesting that changes in snRNP levels affect exon 7 splicing. Altering U1 snRNP activity in cells using a binding-site decoy resulted in a decrease in endogenous SMN2 exon 7 inclusion, suggesting that U1 snRNP abundance affects exon 7 splicing. RNAi-mediated knockdown of SMN or Sm proteins resulted in a decrease in exon 7 splicing. Together, our results support the existence of a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein abundance. These results imply that an increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for understanding therapeutic potential in SMA treatment.
Keywords: RNA and disease, SMN, Spinal muscular atrophy
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein, catalyzing the rearrangement of nucleic acids into thermodynamically more stable structures. It plays role in essentially every step of the retroviral replication cycle. NC is involved in dimerization of the RNA genome, RNA packaging, tRNA primer annealing and various strand-exchange reactions. We previously developed a novel quadruplex formation assay to study the role of NC in facilitating invasion of short DNA duplexes. Other researchers have developed various DNA enzymes (DNAzymes) to cleave RNA in a sequence-specific manner, which could be used in degradation and inactivation of mRNAs of interest. However, DNAzyme activity is limited by stable secondary structures of mRNAs, which should be invaded prior to cleavage. In the present work we investigate role of NC in invasion of nucleic acid secondary structures by complementary sequences. To study NC’s ability to facilitate invasion of DNA quadruplexes, a thrombin binding aptamer, GGTTGGTGTGGTTGG, was investigated in the presence of K+ ions. The reaction was studied as a function of temperature, which revealed that in the absence of NC quadruplex invasion follows a bimolecular pathway with initial partial annealing of the complementary strand. In the presence of NC, the reaction appears to be monomolecular wherein complete unfolding of quadruplex occurs prior to strand invasion. We also studied of invasion of RNA hairpins by DNAzymes and subsequent RNA cleavage. This study showed that in the absence of NC, the DNAzyme is able to invade RNA hairpins with 6-7 bp stems, while in the presence of NC unfolding and cleavage of significantly more stable 10 bp hairpins could be achieved. Taken together, these studies demonstrated that NC has a potential to be used in antisense therapy to facilitate invasion and cleavage of structured mRNA by DNAzymes.
Keywords: HIV-1 nucleocapsid protein, antisense therapy, DNAzymes
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Although U12-dependent introns can be found in most eukaryotic organisms, they represent a small minority of the total number of introns in the human genome. Comparison against U2-dependent introns have yielded results suggesting slower splicing kinetics of U12-dependent introns. This has led some to postulate that these introns might perform regulatory functions on the splicing rates of their host genes. Analysis of these ~600 host genes has revealed some common relationships among their functions, including MAPK signal transduction, cell cycle control and transcription. Because irregularities in these pathways can contribute to unregulated cell growth and abnormalities in androgen mediated cellular activity, we are seeking to determine whether a positive correlation can be drawn between U12-dependent intron regulation and prostate cancer.
Keywords: minor, U12-dependent, introns
Abstract:
Ribosomes are responsible for catalyzing protein synthesis in all cells. Assembly of the four rRNAs and 78 ribosomal proteins in eukaryotic ribosomes requires a large molecular machinery of ~200 proteins and dozens of RNAs. GTPases are one class of assembly factors that is conserved from bacteria to humans and of specific interest due to its potential regulatory role. Previous work has demonstrated that the essential GTPase Bms1 promotes assembly of a pre-40S ribosomal particle essential for rRNA processing.1 Here, we focus on the mechanism by which the GTPase activity within Bms1 is stimulated by an unusual intramolecular GTPase-activating (GAP) domain. We have devised an assay to identify the GAP domain in which GTPase and GAP domains are provided on separate molecules. This assay has identified a 150 amino acid region that provides GTPase stimulation in trans. Interestingly, this region is highly conserved including numerous essential amino acids as well as a conserved phosphorylation site, which regulates the activity of Bms1 in vivo. In vitro analysis of mutant Bms1 has identified two residues that impair GAP activation. The essential amino acids not involved in GTPase activation might be involved in regulating GTPase activity as a result of conformational rearrangements on pre-ribosomes as suggested by previous work.1 Additionally, our experiments indicate the GAP domain is more effective when provided in trans than in cis, indicating the alignment of the domains in full length Bms1 protein is not optimized. We will continue to investigate the interaction of the GAP and GTPase domains in the presence and absence of pre-ribosomes and/or other accessory factors to determine whether these factors affect Bms1’s GAP-stimulated or basal GTPase activity.
References:
1. Karbstein, K., Jonas, S., Doudna, J. (2005) An Essential GTPase Promotes Assembly of Preribosomal RNA Processing Complexes. Mol. Cell 20, 633-643.
Keywords: ribosome assembly, small subunit, GTPase
Abstract:
The ability of cisplatin to serve as a highly effective chemotherapy drug has lead to numerous DNA-binding studies in order to elucidate the drug's mechanism of action. In contrast, research on the interactions of cisplatin with RNA has been very limited. DNA has been considered to be the major target of cisplatin, while RNA has been essentially overlooked. Previous studies in our laboratory demonstrated that RNA is the kinetically preferred target over DNA when similar sizes and structures of nucleic acids are compared. In the present study, we are extending our understanding of cisplatin-binding to the 790 loop of E. coli 16S ribosomal RNA (rRNA). The 790 loop has been shown to be important for ribosomal function, due to its presence in the inter-subunit region and binding site of protein IF3. In addition, this helix from 16S rRNA has been well studied by NMR and mutagenesis experiments. Our current task is to study the binding of cisplatin and various analogues to this small RNA construct and to explore the kinetics of interaction as well as the types and number of adducts formed with this helix. Our studies with full-length 16S rRNA revealed that cisplatin binds preferably to consecutive guanosine residues in helix 24 between positions 799 and 800, while its amino-acid analogues show preference for loop regions containing guanosine and adenosine. We have determined the number of platinum-complexes bound to RNA by using HPLC and atomic absorption spectroscopy, and identified the types of adducts formed by using LC-MS. Compared to mono- and bis-aquated forms of cisplatin, our data suggests that amino-acid derivatives exhibit different binding patterns and activities, which may provide further information towards their usefulness as probes of RNA structure and function.
Keywords: cisplatin, ribosomal RNA, 790 loop
Abstract:
Glucosamine-6-phosphate (GlcN6P) required for cell wall synthesis in all gram-positive bacteria is regulated at the RNA level by the conserved glmS riboswitch. The glmS riboswitch is the only known natural ribozyme that uses GlcN6P as a putative co-enzyme to catalyze RNA cleavage. Co-enzyme dependent RNA cleavage remains an underexplored area in nucleic acid catalysis. We are currently investigating the use of co-enzymes and co-factors by nucleic acid enzymes and are selecting for DNA sequences that will catalyze RNA cleavage with GlcN6P as a co-enzyme. In order to distinguish between a structural versus chemical requirement for GlcN6P in catalysis, we also perform a negative selection with glucose-6-phosphate (Glc6P), which lacks a protonatable putative general acid. Following selections, two pools of DNAzymes, one dependent upon GlcN6P, and the other upon Glc6P, will be obtained. The catalytic mechanism and role of the co-enzyme in the obtained DNAzymes will be investigated.
Keywords: SELEX, DNAzyme, glmS ribozyme
Abstract:
Determining the identity of the catalytic domain of the spliceosome has been a central question in the splicing field for the last two decades. Experimental evidence in vivo and in cellular extracts have indicated that a basepaired complex of U6 and U2 snRNAs in the activated spliceosomes plays a critical role in catalysis of the splicing reaction. In addition, mechanistic and structural similarities to the self-splicing group II introns has led to the widely-believed hypothesis that the spliceosomal RNAs probably form the active site of the splicing reaction, with the spliceosomal proteins playing regulatory and supporting roles. In order to understand the mechanism of how the spliceosomal RNAs affect catalysis, we have successfully reconstituted the catalytic heart of the spliceosome from scratch by assembling the base-paired complex of U6 and U2 snRNAs in vitro. Using in vitro-synthesized, protein free human U6 and U2 snRNAs, we succeeded in forming a U6/U2 base-paired complex that closely resembled the one observed in the activated spliceosomes in terms of its secondary and three-dimensional structure. Intriguingly, we have recently showed that the U6/U2 complex can indeed catalyze a two step reaction resembling the first and second steps of splicing on a pre-mRNA model construct (Valadkhan et al., 2009), thus performing the full spliceosomal catalytic cycle without the help of any proteins in vitro. Using this minimal splicing system, we have studied the effect of several mutations, which disrupt interactions between critical active site elements, on splicing pattern. Interestingly, insertion and deletion mutations in Helix II and Stem I of U2, did not affect the catalytic activity. Even when the whole Helix II and the whole StemI of U2 were deleted, the catalytic activity was unaffected. However, when there were insertion and deletion mutations in the ISL (Intramolecular Stemloop) and Helix I region, we have observed aberrant spliced products. The severity of aberrant splicing was increased when there were mutations in both lower ISL and Helix I. Work is currently underway to determine the effect of similar mutations on splicing in vivo using a orthogonal system in HeLa cells. Taken together these data will help us define the mechanism of splice site selection and the role of snRNAs in specifiying the splice site.
Keywords: U6U2, Splicing
Abstract not available online - please check the printed booklet.
Abstract:
Aminoacyl tRNA synthetases link tRNA molecules correctly to their cognate amino acids for protein biosynthesis. Human lysyl-tRNA synthetase (hKRS) has other functions including its secretion to trigger a proinflammatory response, and its interaction with Gag for uptake and packaging into HIV-1. In our research, NMR studies of the N terminal as well as the anticodon-binding (ACB) domains of hKRS are being studied individually as well as in tandem as a protein referred to here as ‘2D’. These studies are aimed at obtaining a better understanding of how these domains interact with the tRNA. For this purpose, 15N isotopically labeled forms of the N terminal domain, ACB domain and the 2D protein were expressed and purified for NMR study. Titrations of the N terminal, ACB as well as the 2D domains using RNA (oligo U or anticodon stem-loop) were monitored via a series of 1H-15N HSQC experiments. The shifting as well as broadening of numerous protein resonances was observed via RNA titration experiments of the ACB and 2D proteins reflecting the direct interaction of the RNA and ACB domains with each other. NMR studies of the N terminal domain protein indicate that is disordered by itself as well as when it is covalently attached to the ACB domain. The latter domain is much more highly structured by comparison. Further triple resonance experiments conducted upon doubly labeled ACB domain protein will provide site-specific information as far as which portions of this domain are most important for these specific interactions with the RNA. The overall picture of RNA binding by these domains will be presented and discussed.
Keywords: tRNA binding, human lysyl-tRNA synthetase, NMR
Abstract:
Regulation of gene expression by mRNA processing mechanisms wildly exists in eukaryotes, where mRNA polyadenylation and pre-mRNA splicing are two essential steps for the maturation of the most mRNAs. However, much reminds to be learned regarding the regulatory mechanisms of alternative polyadenylation and alternative splicing in plants. Here we focus on a gene in Arabidopsis named OXT6 which has been demonstrated to encode two proteins that may link both polyadenylation and splicing processing. Specifically, intron-2 of the OXT6 gene may play an essential role in the formation of the two different transcripts. Splicing of intron-2 will lead to formation of a transcript encoding a 68 kDa protein, while polyadenylation within intron-2 produces a shorter transcript coding for a 30 kDa proteins. The smaller protein (Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor, AtCPSF30) has been demonstrated to be involved in pre-mRNA polyadenylation, while the larger one contains a domain implicated in mRNA splicing. A set of mutations have been introduced at both the splicing sites and polyadenylation signals within intron-2 to change the splicing and polyadenylation patterns. These constructs are introduced into the wild type and oxt6 mutant backgrounds (where the expression of OXT6 gene is interrupted by T-DNA insertion), and the splicing and polyadenylation events of these constructs will be monitored. From this exercise, the relationships among splicing, polyadenylation, and the different backgrounds may be drawn. The analysis of the expression on OXT6 gene may provide us new evidence on the interaction between splicing and polyadenylation factors in plants, and some basic information on how alternative polyadenylation and alternative splicing affect each other and affect gene expression in general. The results of the research will be presented at the meeting.
References:
Delaney, K., Xu, R., Li, Q.Q., Yun, K.Y., Falcone, D.L. and Hunt, A.G. (2006) Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol, 140, 1507-1521.
Hunt, A. G., Xu, R., et.al and Li, Q. Q. (2008) Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling. BMC Genomics, 9:220
Keywords: mRNA processing, alternative splicing, alternative polyadenylation
Abstract:
tRNAHis guanylyltransferase (Thg1) catalyzes the post-transcriptional addition of an essential G-1 residue to the 5' end of tRNAHis in eukaryotes. Thg1 homologs are found in some bacteria and archaea although the presence of Thg1 is not expected in these organisms due to a genomically encoded G-1. Intriguingly, in vitro activity assays show that bacterial and archaeal Thg1 add residues to the 5' end of tRNA through templated addition, as opposed to the non-templated addition observed in eukaryotes. Consistent with the differences between in vitro biochemical activities exhibited by yeast versus archaeal and bacterial Thg1, the majority of bacterial and archaeal Thg1 homologs will not support the growth of yeast thg1Ä strains with the notable exception of B. thuringiensis Thg1 (BtThg1) and M. smithii Thg1 (MsThg1), suggesting that both may have unique properties distinct from other archaeal and bacterial homologs.
The goal of this work is to further investigate the apparent discrepancy between the in vitro and in vivo activities exhibited by BtThg1 and MsThg1. First the growth rates of the thg1Ä strains containing yeast Thg1, BtThg1, or MsThg1 were compared and shown to be essentially the same, suggesting that BtThg1 and MsThg1 have the ability to act as efficiently as yeast Thg1 in vivo, despite their different in vitro activities. Second, tRNAs isolated from the same strains using a hot phenol method were probed for the presence of a G-1 using primer extension assays specific for tRNAHis; these assays showed that a single nucleotide was being added at the –1 position in all three strains. Unexpectedly, U-1 was also being incorporated into tRNAHis in vivo. Further assays will be done to determine if there are differences in the relative quantities of U-1 and G-1 addition and whether nucleotides are added to other tRNA substrates in vivo in any of these strains.
Keywords: yeast, tRNAHis, Thg1
Abstract:
The removal of nuclear pre-mRNA introns by splicing is crucial to the expression of most genes in higher eukaryotes. The U12-dependent spliceosome catalyzes the removal of the less abundant class of U12-dependent introns. Despite their low abundance, U12-dependent introns are found in both tissue specific and ubiquitously expressed genes, some of which carry out essential cellular functions such as DNA replication and repair, transcription, RNA processing, and translation. Accordingly, the correct identification and excision of minor class introns by the U12-dependent spliceosome is essential. This study seeks to investigate the structure-function relationship of the U11 snRNA, a small nuclear RNA that functions in U12-dependent pre-mRNA processing. Because the U11 snRNP resembles the U1 snRNP in the U2-dependent spliceosome, it is thought to be the analogue of U1 for splicing in this rare class of introns. We have developed a U11 mediated 5’ splice site triple mutation suppressor assay for a U12-dependent intron. Using this suppressor system, currently we are generating a large number of U11 snRNA suppressors to identify functionally relevant regions.
Keywords: Splicing, suppressor assay, U11 snRNA
Abstract not available online - please check the printed booklet.
Abstract:
Structured RNA molecules fold hierarchically to form complex, protein-like 3D structures. Their function depends on forming the correct 3D structure. Unlike DNA, RNA molecules are single-stranded. The secondary structure of an RNA consists, therefore, of short double-helices that form when the RNA strand folds back on itself so that Watson-Crick complementary regions can base-pair. Once the sequence of an RNA is determined, programs such as Mfold (Zuker, 2003) are used to predict its secondary structure. Typically, about 2/3 of the bases of an RNA form Watson-Crick helices. The rest of the bases appear in secondary structures as unpaired “loops” – hairpin loops, which occur at the end of a helix, internal loops, between two helices, or junction loops, connecting more than two helices. However, X-ray crystallography and NMR spectroscopy have shown that most of these RNA “loops” are actually well-structured 3D motifs, consisting of one or more non-Watson-Crick basepairs (Leontis & Westhof, 2001) and responsible for important biological functions. Some of these 3D motifs mediate tertiary interactions that stabilize the 3D fold of the RNA. Other motifs bind proteins or small molecules, including drugs and ions. Others interact with other RNAs. Many of these “loop” motifs are recurrent – they are found in many different, unrelated RNAs, where they form very similar 3D structures and play similar functional roles. Thus, it is desirable to predict the 3D structures of hairpin, internal, and junction motifs based on their sequences. Different instances of the same 3D motif can differ in sequence and in number of nucleotides, due to substitutions, insertions or deletions of bases at specific positions. To predict instances of a motif from sequences, it is necessary to understand the possible sequence variations of each motif – this is what we call the sequence signature of the motif. To define the sequence signature of each motif, we search a reduced-redundancy set of atomic-resolution RNA structure files from the Protein Data Bank (PDB) using the “Find RNA 3D” (FR3D) software package (Sarver, Zirbel, Stombaugh, Mokdad, & Leontis, 2008). Motifs that share the same structural features as query motifs are stored in a library and are used to build probabilistic models based on stochastic context-free grammars.
References:
1. Leontis, N. B., & Westhof, E. (2001). Geometric nomenclature and classification of RNA base pairs. RNA, 7(4), 499-512.
2. Sarver, M., Zirbel, C. L., Stombaugh, J., Mokdad, A., & Leontis, N. B. (2008). FR3D: finding local and composite recurrent structural motifs in RNA 3D structures. J Math Biol, 56(1-2), 215-252.
3. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res, 31(13), 3406-3415.
Keywords: RNA 3D Motifs, RNA structure, Find RNA 3D program
Abstract:
tRNAOther is a tRNA-like small RNA of unknown function in Bacillus cereus that is aminoacylated in vitro by the concerted effort of unrelated class I and II lysyl-tRNA synthetases (LysRS). In vivo analyses revealed that class I LysRS and tRNAOther were mainly produced during stationary phase, while class II LysRS was produced in both exponential and stationary phases. tRNAOther could not be detected in polysomes isolated from cells collected during exponential or early stationary phases, indicating a function beyond translation. tRNAOther is encoded within a pathogenicity island together with several putative virulence factors. To investigate the role of tRNAOther in B. cereus, we deleted the corresponding gene and screened for changes in growth phenotype compared to wild-type. Deletion of tRNAOther was not deleterious, and growth in rich media and ability to sporulate were virtually unchanged. Changes were observed in stationary phase, such as de-regulation of the production of a bacteriocin-like inhibitory susbstance and increased responsiveness to various germinants. Extensive screening for growth phenotype changes (~1900 conditions) was carried out by performing phenotypic microarray analyses (BIOLOG, Inc.). These studies revealed significant changes; B. cereus ΔtRNAOther gained resistance to several antibiotics, but also acquired sensitivity to a number of compounds including antibiotics, cationic detergents and positively charged ionophores. Changes in gene expression between the wild-type and ΔtRNAOther strains were explored by transcriptional microarrays. Surprisingly, almost no changes in transcript levels were observed between wild-type and ΔtRNAOther strains collected during stationary phase. In contrast, many significant transcript level changes were observed between wild-type and ΔtRNAOther strains collected during mid-exponential growth. Among these, changes were observed for several transcriptional regulators, ABC transporters, and genes involved in sporulation and germination. The function of tRNAOther as a regulatory RNA is currently being investigated.
Keywords: tRNA, small RNA, LysRS
Abstract:
The goal of this research is to examine ribosome assembly defects using mass spectrometry-based methods. The aim of this project is to use mass spectrometry to identify the post-transcriptional RNA modifications and post-translational ribosomal protein modifications that are unique or absent from improperly folded ribosome assembly subunits.
Escherichia coli is being used as the model system. Two strains of E. coli (K-12 and SK5665) are grown in the presence and absence of erythromycin at 25 oC and 37 oC. Subunits, intermediate, and intact 70S are separated and isolated by use of sucrose fractionation. Post-transcriptional modifications to RNA are identified using liquid chromatography with ultraviolet absorption (LC-UV) and mass spectrometry (LC-MS). Ribosomal proteins are identified, and any post-translational modifications confirmed, using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS).
When erythromycin sensitive cells are cultured in the presence of erythromycin, the 50S subunit is unable to properly form, resulting in an intermediate 50S particle which sediments between 30S and 50S. The assembly particle is composed of both 5S and 23S rRNA and an undetermined number of large subunit ribosomal proteins. In wild-type cells, this mis-folded particle is degraded by the RNase E complex. E. coli strain SK5665 cells contain a temperature-sensitive RNase E phenotype, and RNase E can be inactivated by culturing at 25 oC, thus allowing this mis-folded 50S intermediate to be accumulated and isolated by sucrose fractionation. Currently, we have been characterizing the rRNAs and ribosomal proteins present in this mis-folded 50S intermediate and comparing their identity and modification status to the rRNAs and ribosomal proteins found in properly assembled 50S subunits. These data will provide insight into differences at the primary structure level between ribosomal subunits that can correctly fold with those that do not fold correctly.
Keywords: ribosome assembly, rRNA, liquid chromatography-mass spectrometry
Abstract:
Modified nucleosides in RNA are formed post-transcriptionally by modifying enzymes. The functional significance and structural role of modified nucleosides in ribosomal RNA (rRNA) and in transfer RNA (tRNA), remain poorly understood except for the tRNA anticodon region. These modified nucleosides are important in stabilization of the tertiary structure of tRNA and are essential to proper decoding (codon/anticodon interactions). Modification levels of tRNA have been shown to increase with increasing culture temperature and post-transcriptional modification in tRNA contributes to thermal stabilization in hyperthermophilic archea. The transcription of at least one tRNA modifiying enzyme, miaA, has also been found to increase during heat shock. The heat shock response is a mechanism which allows a cell to cope with a sudden increase in temperature or other environmental stress. We are interested in using the selectivity and sensitivity of LC-MS based techniques to investigate the changes in global modification patterns in rRNA and tRNA as the result of heat shock. Investigating how post-transcriptional modifications change in response to cellular stress may help us to understand the functional significance of these modifications. The relative amounts of modified nucleosides present in RNA isolated from heat shocked and normal E. coli cell cultures will be presented.
Keywords: tRNA, modified nucleosides, heat shock
Abstract not available online - please check the printed booklet.
Abstract:
Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively.
Keywords: Bacteriophage phi29, pRNA, gene delivery
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The pRNA (packaging RNA) of bacteriophage phi29 has been demonstrated having a novel role in gearing the phi29 DNA packaging motor and in delivering therapeutic RNAs for the treatment of cancers, viral infections, and genetics diseases. Since RNA is vulnerable to RNase and can be easily degraded in vitro and in vivo, approaches with chemical modification have been extensively explored in the quest of reliable methods to produced stable RNA. One critical question remains to be answered is whether the RNA maintain its correct folding and biological activity after chemical modification. Here we evaluate the property of 2¡¯-Fluorine (2¡¯-F), 2¡¯-Amino (2¡¯-NH2), 2¡¯-O-Methyl (2¡¯-O-Me), and modified RNA using the highly sensitive phi29 DNA virion assembly system. It was found that with the 2¡¯ modification on only pyrimidines but not purines, the resulting RNA was completely resistant to RNase digestion and remains intact even after 19 hours of incubation with fetal bovine serum. Moreover, pRNA with 2¡¯F modification retained dimer formation potential via loop-loop interaction. More interestingly, the 2¡¯F modified pRNA retained its activity in binding to the procapsid, gearing the motor to package the genomic DNA, and retained its property in the process of assembling infections phi29 virion. 2¡¯-NH2 modification on pyrimidines also resulted in RNase resistant pRNA, however, the resulted pRNA could not form dimers, and was incompetent in viral assembly. For the 2¡¯-O-Me modification, pRNAs could be synthesized through in vitro transcription with 2¡¯-O-Me modified cytidines (2¡¯-O-Me-C) as substrate, while synthesis was failed using 2¡¯-O-Me modified uridines as substrate. pRNA with 2¡¯-O-Me-C modification was competent in pRNA dimer formation and procapsid binding. However, the modified pRNA were inactive in phi29 virion assembly. The pRNA containing 2¡¯-O-Me-C was not as stable as pRNA with 2¡¯-F and 2¡¯-Amino pyrimidines modification. Taken together, our data suggested that 2¡¯-F modification did not affect pRNA folding or hinder its biological activity. However, 2¡¯-O-Me and 2¡¯-NH2 modification is seriously detrimental to RNA folding, structure and function.
Keywords: Bacteriophage phi29, RNA modification, pRNA
Abstract:
U12 snRNA binds to the branch site sequence of U12-dependent spliceosomal introns by Watson-Crick base pairing and is essential for splicing. Although much is known about U12 snRNA region which base pairs with the branch site of minor class introns, virtually nothing is known about the functional role of other regions of the snRNA. Here, we examined in vivo requirement of stem IIa, stem IIb, stem III and single stranded regions of U12 snRNA using a previously described U12 snRNA mediated branch site mutation suppression assay. Mutations of single stranded linker region immediately following the stem I region revealed that distance constraint between stem I and stem IIa is necessary for splicing. Various mutations of stem IIa including complete deletion of the stem loop were deleterious for in vivo splicing. Surprisingly, mutations of the evolutionarily conserved stem IIb including complete deletion have little or no functional effect. Finally, we show the requirement of stem III terminal loop for in vivo splicing. Interestingly, deletion designed to shorten the length of stem III was functional. Our mutagenesis study of stem III supports the alternative version of the stem loop structure proposed recently. These results show that in vivo sequence requirements of U12 snRNA are similar to those described previously for U2 snRNA and provide experimental support for U12 snRNA intramolecular structure.
Keywords: U12 snRNA, suppression assay, splicing
Abstract:
Prostate cancer (PCa) is the most common type of cancer found in American men, and the second leading cause of cancer related illness and deaths in the United States. Recent epidemiological study shows that 1 in every 6 men over the age of 45 is at risk of PCa. Androgen receptor (AR) plays a causative role in the development of hormonal-refractory PCa. Hormonal blockade therapy which inhibits the expression of AR eventually fails and disease progresses to fatal androgen-refractory stage from androgen-dependent stage. Therefore, novel molecular approaches which can target and block the expression of AR are urgently required. We propose that microRNAs (miRNA) that function as negative gene regulators have potential as PCa therapeutics. Using bioinformatics methods we have identified that human miRNA hsa-miR-E has the potential to inhibit AR expression. In the present study we are carrying out experiments to validate AR as a target of miR-E. Our preliminary data show that miR-E can suppress the expression of AR in prostate cancer cells; currently we are testing the effect of miR-E overexpression and resulting AR suppression on the growth and proliferation of prostate cancer cells.
References:
1.Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004 Jan 23;116(2):281-97
2.Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer. 2006 Nov;6(11):857-66. Calin GA,
3.Croce CM. MicroRNA-cancer connection: the beginning of a new tale. Cancer Res. 2006 (15):7390-4
Keywords: miRNA, prostate cancer
Abstract:
tRNAHis guanylyltransferase (Thg1) is an essential enzyme in yeast that adds a single G residue at the -1 position (G-1) of tRNAHis, causing extension of the tRNA in the 3'-5' direction. G-1 is a necessary recognition element for HisRS and is conserved among nearly all tRNAHis species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown. Thg1-catalyzed G-1 addition occurs via a complex mechanism involving three steps: adenylylation, nucleotidyltransfer, and removal of the 5' pyrophosphate. A complete understanding of this complex reaction mechanism requires isolation and characterization of each of these catalytic steps individually. Our investigations reveal three intriguing observations. First, using transient kinetic assays we have shown that the first order rate constants for nucleotidyltransfer (kntrans) and removal of pyrophosphate (kppase) increase with increasing pH (6.0-7.5) while the rate constant for adenylylation (kaden) decreases, implying different ionization event(s) for different steps of the Thg1 reaction, and suggesting the likelihood of identifying residues that function independently for each step. Second, despite the fact that there is no obligate role for NTPs as substrates for the pyrophosphatase step, the presence of GTP increases kppase significantly suggesting previously unsuspected role(s) for nucleotides in this reaction. Third, in the presence of Mn+2 at pH > 8.0 we have detected evidence indicative of an adenylylated enzyme intermediate not previously observed. Adenylylated and guanylylated enzyme intermediates are a common feature in the mechanism of ligase and mRNA capping enzymes which also activate polynucleotide substrates via a 5'-5' phosphoanhydride bond.
Keywords: tRNAHis, guanylyltransferase, nucleotidyltransfer
Abstract:
Aminoacyl-tRNA synthetases attach specific amino acids to cognate transfer RNAs (tRNAs), an essential step in accurate decoding of genetic information. Prolyl-tRNA synthetases (ProRSs) are known to mischarge tRNAPro with the smaller amino acid Ala, and with Cys, which is the same size as Pro. Quality control in translation is partly ensured by an editing domain (INS) present in most bacterial ProRSs that hydrolyzes Ala-tRNAPro but not Cys-tRNAPro. The latter is cleared by a free-standing INS domain homolog, YbaK. To understand the molecular basis of the distinct substrate specificities of these homologous editing domains, we have investigated the mechanism of hydrolysis of Cys-tRNAPro by YbaK. Our data support a substrate-assisted mechanism of catalysis wherein hydrolysis is mediated by the substrate side chain thiol. We have previously reported that YbaK and ProRS interact in vitro. In new work, we now show that the ProRS·YbaK complex demonstrates enhanced proofreading activity relative to the YbaK protein alone. The complex competes efficiently with Ef-Tu for Cys-tRNAPro, whereas YbaK alone does not, implying that YbaK functions before the substrate is released from ProRS. To probe protein-protein interactions in vivo, a split-GFP reassembly technique was employed and results support our hypothesis that ProRS, YbaK, and tRNA form a complex in the cell. Taken together, we propose that the ProRS INS and YbaK domains interact in vivo and function via distinct mechanisms involving steric exclusion and thiol-specific chemistry, respectively, thus collaborating to ensure accurate decoding of Pro codons.
Keywords: YbaK, aminoacyl-tRNA synthetase, substrate-assisted catalysis
Abstract:
Ribosome assembly requires binding and release of accessory proteins from pre-ribosomes. For different accessory factors to stably associate with and dissociate from pre-ribosomal complexes at specific stages, energy must be input during binding or dissociation to alter the free energy of the protein-complex interactions. Fap7 is an accessory factor essential for small subunit (SSU) assembly and has sequence homology to ATPases. Mutations in conserved amino acids predicted to be involved in Fap7’s ATPase activity are essential for growth and ribosome assembly in yeast (1). Previous work has shown that Fap7 interacts directly with the SSU component RpS14 and depletion of Fap7 leads to retention of Rio2, a protein kinase required for 20S processing, in pre-ribosomal complexes (1). Our own experiments indicate that this retention is specific and not simply due to increased levels of 20S rRNA because Nob1, which also binds this intermediate, does not accumulate when Fap7 is depleted. Based on these data, we propose the following model: Rio2 bound to the pre-ribosome inhibits cleavage of the 20S pre-rRNA to produce the mature 18S rRNA. Fap7 binds Rps14 in proximity to Rio2 and uses its ATPase activity to remove Rio2 from pre-ribosomes, either through phosphorylation of Rio2, another protein, rRNA, or through a non-kinase motor-like ATPase activity, allowing continuation of ribosome maturation. We are using a combination of biochemical techniques and in vivo studies to explore Fap7’s essential role in ribosome assembly.
References:
Granneman et al. Mol Cell Biol. 2005 25(23): 10352-64.
Keywords: rRNA processing, ribosome assembly
Abstract not available online - please check the printed booklet.
Abstract:
Trm10 is a highly conserved SAM-dependent methyltransferase which catalyzes N1-methylation at the G9 position of tRNA substrates. While all m1G9 methylation is catalyzed by a single TRM10 in yeast, there are up to three Trm10 homologs in metazoa, including humans, for which the function has not yet been investigated. In humans, one of these homologs (RG9MTD1) is targeted to the mitochondria where it has unexpectedly been implicated in a novel proteinacious RNase P complex. Although the biochemical function of Trm10 in this complex is unknown, its presence is absolutely required for RNase P processing activity. Furthermore, the function of the remaining two, presumably cytoplasmic, homologs (RG9MTD2 and RG9MTD3) is unknown. Thus, the goal of this work is to investigate the biochemical and biological function of the multiple human Trm10 homologs.
To that end we have begun to characterize one of the human Trm10 homologs, RG9MTD2, which shares the highest sequence homology to yeast Trm10. Using site specifically-labeled yeast Gly, Val and Leu tRNAs and assay conditions optimized with respect to [Mg] and pH, we have shown that RG9MTD2, like yeast Trm10, catalyzes m1G9 formation. In addition, RG9MTD2 follows the same pattern of in vitro activity observed with yeast Trm10, modifying Gly and Val but not Leu tRNAs, which is unexpected since a different subset of tRNAs are m1G9 modified in yeast versus human cells. Previously, similar questions of substrate specificity have been raised regarding yeast Trm10, which by some unknown mechanism modifies some, but not all, G9-containing tRNAs in vivo. Therefore, the basis for Trm10 substrate specificity in both yeast and humans is unclear, and future investigations of this issue, as well as similar investigations of the remaining two human homologs (RG9MTD1 and RG9MTD3) are in progress.
Keywords: tRNA methyltransferase, yeast, enzyme catalysis
Abstract not available online - please check the printed booklet.
Abstract:
Conformational changes are important in RNA for both binding and catalysis. We are developing computational methods for exploring and understanding pathways for defined conformational changes.
One system of study is the conformational change of a non-canonical pair. In an NMR structure of an AA mismatch in the sequence:
5’ GGUGAAGGCU3’
3’PCCGAAGCCG 5’
(P = purine), the AA non-canonical pair is in conformational exchange between a minor and major conformations. The conversion of the major trans Hoogsteen-sugar to the minor trans sugar-Hoogsteen non-canonical pair occurs on the NMR timescale.
We used the AMBER molecular mechanics software package to model conformational change pathways. Nudged Elastic Band (NEB) was used to predict minimal potential energy paths using a series of all atom images of the system along the path. Both TMD and NEB provided insight into conformational change pathway. NEB provided a time-independent and discrete low potential energy pathway.
Predicted pathways from NEB were analyzed and a reaction coordinate determined for the conformational change. This reaction coordinate involved an improper dihedral angle defined by C8, C4, and N1 on one adenine and C5 on the second adenine in the non-canonical pair. The minor state had an improper dihedral value of about 0 degrees, while the major state had a value of about 180 degrees. Umbrella sampling was then used to predict the free energy profile along the 360 degree reaction coordinate. Umbrella sampling was done using 36 windows of 10 degrees each with 12 ns of sampling per window for 6 different random number seeds. Total sampling involved about 2.6 microseconds of MD spanning about 7 total years of CPU time. The free energy profile suggested errors in the AMBER force field because there was a reversal in the relative free energies of the major and minor structures. Investigation of a putative stacked state identified in the free energy profiles indicated the force field overestimated stacking stability. Revaluation of the free energy profile via QM/MM calculations with PM3-PDDG improved the free energy profile.
References:
1. Chen, et al. 2006. An alternating sheared AA pair and elements of stability for a single
sheared purine-purine flanked by sheared GA pairs in RNA. Biochem. 45:6889-6903
2. Case, et al. 2005. The AMBER Biomolecular Simulation Programs. J. Comp. Chem. 26: 1668-1688
3. Mathews & Case. 2006. Nudged elastic band calculation of minimal energy paths for the
conformational change of a GG non-canonical pair. JMB. 357:1683-1593
4. Torrie & Valleau. 1977. Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling. J. Comp. Phys. 23:187-199
5. Kumar, et al. 1992. The Weighted Histogram Analysis Method for Free Energy Calculations of Biomolecules. J. Comp. Chem. 1992. 13:1011-1021
6. Walker, et al. 2008. The Implementation of a Fast and Accurate QM/MM Potential Method in Amber. J. Comp. Chem. 29:1019-1031
Keywords: Molecular Mechanics, RNA Conformational Dynamics, Conformational Change Pathways
Abstract not available online - please check the printed booklet.
Abstract:
The Hu RNA-binding protein family consists of four members: HuR/A, HuB, HuC and HuD. HuR expression is widespread. The other three neuron-specific Hu proteins play an important role in neuronal differentiation through modulating multiple processes of RNA metabolism. In all of the splicing events examined, Hu proteins promote skipping of the alternative exons. Here we report the first example where Hu proteins promote inclusion of an alternative exon, exon 6 of the HuD pre-mRNA. Sequence alignment analysis indicates the presence of several conserved AU-rich sequences both upstream and downstream to this alternatively spliced exon. We generated a human HuD exon 6 mini-gene reporter construct that includes these conserved sequences. Hu protein over-expression led to significantly increased exon 6 inclusion from this reporter and endogenous HuD. Studies using truncated and mutant HuD exon 6 reporters demonstrate that two AU-rich sequences located downstream of exon 6 are important. RNAi knockdown of Hu proteins decreased exon 6 inclusion. An in vitro splicing assay indicate that Hu proteins promote HuD exon 6 inclusion directly at the level of splicing. Our studies demonstrate that Hu proteins function as splicing enhancers and expand the functional role of Hu proteins as splicing regulators.
Keywords: alternative splicing, HuD
Abstract not available online - please check the printed booklet.
Abstract:
RNase P, a universal and essential catalytic ribonucleoprotein responsible for cleaving the 5'-leader of precursor tRNA transcripts, has been successfully reconstituted from several different archaeal representatives. Although both types of archaeal RNase P—types A and M, which are similar to bacterial and eukaryal RNase P, respectively—have been reconstituted, all archaeal type A holoenzymes studied to date are thermophilic in origin and require high temperatures (> 55 oC) for optimal pre-tRNA processing activity. The availability of a mesophilic archaeal RNase P (such as the one from Methanobrevibacter smithii) will permit single molecule FRET and rapid quench flow kinetic studies, thus far not possible due to the technical limitations imposed by the optimal temperature for functioning of the thermophilic archaeal RNase P variants. Other payoffs from studies on M. smithii RNase P are likely. Since M. smithii, the predominant archaeon in the human gut, is a methanogen whose syntrophic relationship with saccharolytic bacteria permits increased fermentation of sugars in the digestive process and ultimately a greater caloric absorption from food, it is viewed as a potential anti-obesity target. As the subunit makeup of archaeal RNase P differs from its eukaryal and bacterial relatives, it might be possible to design a specific inhibitor of M. smithii RNase P without harming the animal host or commensal bacteria. Towards the goal of reconstituting the M. smithii RNase P holoenzyme for biochemical characterization and screening of small-molecule inhibitors, the genes encoding the single RNA and protein subunits have been cloned into expression vectors. We will present data from our ongoing efforts to (i) purify recombinant versions of the individual subunits [subsequent to in vitro transcription of the RNA or overexpression of the proteins in E. coli BL21(DE3)], and (ii) reconstitute a functional holoenzyme in vitro from its constituent subunits.
Keywords: RNase P
Abstract:
In this study, we have used fluorescence resonance energy transfer (FRET) and single molecule spectroscopy to analyze the effects that 2’ backbone modifications have on the dynamics of molecular beacons (MB). Molecular beacons are advantageous for live cell assays because they only fluoresce in the presence of the target molecule, negating the need to wash away unbound probes, which allows delivery into living cells. Molecular beacons with 2’ backbone modifications are expected to prevent nuclease degradation in the cells. 2’F, 2’OMe, and 2’H modifications were characterized and compared to the parent 2’OH MB via steady state, time resolved, and single molecule FRET under physiological conditions. These studies indicate that the modified MBs act similarly to the 2’OH MB. The investigated MBs bind with similar affinity to the RNA target. The 2’OMe MB binds with 3-fold better affinity than the 2’OH MB, while the 2’F binds with similar affinity and 2’H MBs binds with 3-fold less. Single molecule studies show a lack of MB dynamics observed in the minute timescale, as well as a large dynamic range in that the molecular beacon alone was primarily in the closed conformation and, in the presence of the target, the beacon was primarily in the open conformation.
Keywords: molecular beacon, single molecule FRET
Abstract:
Ribosomes catalyze protein synthesis in all cells and as such are among the most important biomolecules. In eukaryotes assembly of ribosomes requires >200 RNA and protein cofactors, whose function remains poorly understood. In S. cerevisiae, the final cytoplasmic step in the maturation of the 40S (small) subunit involves cleavage of the 20S rRNA precursor to the final 18S product. Several accessory factors are involved in this step including two proteins Nob1 and Dim2 (1,2). While Nob1 is the endonuclease responsible for cleavage of the 20S rRNA (1, 3, 4) the function of Dim2 remains unknown. Previous research suggested an interaction between Nob1 and Dim2 (5). Sequence alignments suggest that Dim2 contains three putative KH domains. Various truncations of full length Dim2, including deletion of the N-terminal and C-terminal regions, were cloned and purified. As shown previously (6), our data implicate the C-terminal KH domain in rRNA binding. Preliminary analysis by size exclusion chromotagraphy shows that the N-terminal region is responsible for dimerization of Dim2. Initial pull-down data indicate that the middle domain of Dim2 is likely responsible for binding Nob1. A model of Dim2 suggests W113 and K115 as candidates for residues involved in the interaction with Nob1. We are currently testing the role of these residues in protein-protein binding. Once the Dim2-Nob1 interaction has been detailed in vitro, we will determine the effect of this interaction on ribosome assembly in vivo.
References:
1. Fatica, A., Oeffinger, M., Dlakic, M., and Tollervey, D. Mol and Cell Biol. 2003; 23: 1798.
2. Senapin, S, Clark-Walker, G. D., Chen, X. J., Serapin, B., and Daugeron, M. Nucleic Acids Res. 2003; 31: 2524.
3. Fatica, A., Tollervey, D., and Dlakic, M. RNA. 2004; 10: 1698.
4. Lamanna, A. and Karbstein, K. PNAS. (2009) submitted for publication.
5. Tone, Y., and Toh-e, A. Genes Dev. 2002; 16: 3142.
6. Vanrobays, E., Leplus, A., Osheim, Y. N., Beyer, A., Wacheul, L., and Lafontaine, D. RNA. 2008; 14: 2061.
Keywords: Ribosome Assembly, Dim2, KH domain
Abstract not available online - please check the printed booklet.
Abstract:
Computational docking of small molecules to RNA may soon be a key concentration in drug discovery. As a result of its highly-conserved primary and secondary sequence elements, the T box riboswitch, which regulates gene expression in many Gram-positive bacteria, is a potential target for pharmacological therapeutics. The riboswitch consists of the leader region of RNA that, depending upon environmental factors, can form either an antiterminator or terminator structure, thus continuing or terminating transcription. By establishing a technique to signal the terminator structure to be formed (i.e. introducing a small molecule), certain processes that are necessary for protein biosynthesis could be halted. In the current work, docking experiments were performed with Autodock 4.2. Autogrid 4.2 and AutoDockTools 1.5.4 were used to ready the ligands and the receptor for docking. In these docking experiments, classes of small molecules docked with an antiterminator model RNA, AM1A, were oxazolidinones and triazole compounds. The ligands were chosen based on fluorescence anisotropy screening data and structural components. All ligands were docked in the unprotonated and monoprotonated forms. While a variety of docking sites were observed, many docked conformations shared a common binding site in AM1A. Based on results from the fluorescence anisotropy data, the ligands were categorized by their likelihood to bind to the RNA. Calculations were performed to find average binding energies of these ligands bound to the AM1A, and trends were observed that supported the previous fluorescence data.
Keywords: riboswitch, small molecules, docking
Abstract:
Ribosomes catalyze protein synthesis in all cells. Even though assembly of ribosomes is well studied and understood in bacteria, assembly in eukaryotes is much more complex and requires >170 accessory proteins. These proteins facilitate processing and folding of the four rRNAs and their assembly with the 78 ribosomal proteins, most of which have no prokaryotic homologs. However, the exact function of most ribosome assembly factors remains unknown. Three of the four rRNAs, the 18S, 5.8S and 25S rRNAs, are co-transcribed in a single transcript, which is then cleaved in a series of well-ordered steps to release the mature rRNAs. While the spatial and temporal ordering of these cleavage steps has been determined, the mechanisms by which they occur have yet to be elucidated.
Data in our lab suggest that prior to and immediately after cleavage at site A2 (which separates 18S rRNA from the 5.8S and 25S rRNA), the rRNA is in a conformation different from that required for the last cleavage step. In order to achieve this final structure, RNA must be removed after A2 cleavage. Our model predicts that the interaction between two essential assembly factors, Rrp5 and Rok1, is responsible for this conformational change. The c-terminus of Rrp5, an rRNA binding protein, has been shown to be essential for A2 cleavage. Additionally, Rrp5 has been shown to have both a physical and genetic interaction with Rok1, a putative DEAD-box RNA helicase.1,2 We hypothesize that Rok1 interacts with the rRNA via Rrp5 and uses its ATPase activity to facilitate the essential rRNA conformational change.
Preliminary northern analysis results suggest that Rok1 depleted cells accumulate the expected 18S rRNA precursors. In biochemical analyses of Rok1, ATPase inhibition assays in the presence of AMPPNP or ADP indicate that Rok1 binds ADP at least 500 fold tighter than ATP. In vivo, an accessory protein such as Rrp5 may be required for increased ATP affinity and Rok1 activation. In further support of the Rok1-Rrp5 interaction, pull-down experiments with recombinant proteins show that Rok1 binds Rrp5 directly.
Future directions include development of an in vitro helicase assay using the expected in vivo duplex substrate, Rok1 and Rrp5.
References:
1 Torchet, C.; Jacq, C.; Hernann-Le Denmat, S. RNA 1998, 4, 1636.
2 Vos, H.R.; Bax, R. et al. Nucleic Acids Research 2004, 32, 5827.
Keywords: Ribosome Assembly, 18S rRNA Maturation, Rok1 and Rrp5
Abstract:
The results of the ENCODE project suggest that while over 93% of the human genome is transcribed into RNA, ORFs and their associated UTRs occupy only 2% of the genome (Birney et al., 2007; Mattick, 2007). Interestingly, the percentage of non-coding to protein-coding genomic sequences is much greater in higher eukaryotes compared to simpler organisms, suggesting that the non-coding sequences might be involved in the evolution of complexity (Taft et al., 2007). It is estimated that human genome contains over 70,000 large non-protein coding transcripts (lncRNAs); however, the functional mechanism of this novel class of cellular regulators is almost completely unknown. We have analyzed BORG RNA, a 2766 nucleotide long transcript originally discovered in mouse myoblasts which have been shown to contain no protein-coding capability (Takeda et al., 1998). BORG contains five repeat elements and a region that is ~65% conserved from mouse to human. We have shown that BORG is highly expressed in neural tissues in mouse, as well as in primary cultured neurons, but not astrocytes or oligodendrocytes. We made stable cell C2C12 lines overexpressing full-length BORG and induced muscle differentiation to determine if BORG overexpression affects their ability to differentiate. Surprisingly, the cells developed round cell bodies and long branched processes that interconnected to form a network, features characteristic of neuronal cells. Both immunostaining and RT-PCR data show that the BORG overexpression cells express different neuronal markers in a timely regulated way along their neuronal differentiation. To further characterize the mechanism of this neuronal differentiation, we made different truncation mutations of BORG by deleting the repeat elements one at a time, and overexpressing the truncated RNAs in C2C12 cells. Interestingly, some mutants totally lost the ability of neuronal differentiation while others could still differentiate. This study is a first step toward characterizing the mechanism by which a large non-coding RNA can reprogram muscle precursor cells, which are of mesodermal origin, to neuronal cells, which are of ectodermal origin. Taken together, our study provides an example of the crucial, and hitherto unknown, roles played by non-coding RNAs in regulation of cellular function and development.
Keywords: repeat elements, large non-protein-coding RNA, lncRNA
Abstract:
Bacteriophage phi29 DNA-packaging motor is geared by six packaging RNAs (pRNA). The pRNA molecules have been reported to serve as building blocks in RNA nanotechnology, and as vehicles for specific delivery of therapeutics to treat cancers and viral infections. The understanding of the 3D structure of pRNA and its location and positioning on the motor are both fundamentally and practically important. A customized single-molecule dual-color imaging system has been constructed to study the structures of pRNA molecules. The system is the combination of a low-temperature (-80 ¡ãC) sensitive electron multiplied CCD camera and the prism-type total internal reflection mechanism. A laser combiner was introduced to facilitate simultaneous dual-channel imaging. It has been applied to study the structure, stoichiometry, distance and function of the phi29 DNA packaging motor. Single molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of pRNA on the motor and elucidated the mechanism of pRNA hexamer assembly. The feasibility of the single-molecule imaging system was demonstrated in studies of single-molecule FRET. Distance rulers made of dual-labeled dsDNA and RNA/DNA hybrids were used to evaluate the system by determining the distance between one FRET pair. The single-molecule FRET was also applied to the reconstructed the 3D structure of phi29 motor pRNA monomers and pRNA dimers. Ten pRNA monomers labeled with single donor or acceptor fluorophore at various locations were constructed, and eight partner pairs were assembled into dimers. FRET signals were detected for six dimers and utilized to assess the distance between each donor/acceptor pair. The results provide the distance constraints for 3D computer modeling of phi29 DNA packaging motor. We have also re-engineered the energy conversion protein, gp16, of phi29 motor for single fluorophore labeling to facilitate the single molecule studies of motor mechanism. The potential applications of single-molecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with co-localization (SHREC) approaches to the study of the phi29 nanomotor were also investigated.
Keywords: Bacteriophage phi29, Dual-channel imaging, Single molecule imaging
Abstract:
In vivo, many RNA molecules can adopt multiple conformations depending on their biological context. For example, an RNA molecule that is initially in a stable hairpin conformation will at a later stage of its biological cycle interact with a second RNA molecule, which in turn will trigger a dimerization reaction of the two molecules. This is the case of the HIV Dimerization Initiation Sequence (DIS) or the DsrA RNA in bacteria. It is quite common that the initial interaction between the two RNAs takes place via complementary unpaired regions, thus forming a so-called kissing complex. However, the exact kinetic mechanism by which the two RNA molecules reach the dimerized state is still not well understood.
To investigate the refolding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with laser induced temperature jump kinetics, called Laser Assisted Single-molecule Refolding (LASR). LASR enables us to induce folding reactions of otherwise kinetically trapped RNAs at the single molecule level, and to characterize their folding landscape. Single molecule time trajectories show that we can drive the dimerization reaction between two trapped kissing RNA hairpins with LASR, and use this data to calculate folding enthalpies and entropies. LASR provides an exciting new approach to study molecular memory effects and kinetically trapped RNAs in general. LASR should be readily applicable to study DNA and protein folding as well.
Keywords: single molecule, FRET, temperature jump
Abstract not available online - please check the printed booklet.
Abstract:
The T box transcription antitermination riboswitch regulates gene expression by interaction of uncharged cognate tRNA with the 5′ untranslated region of the mRNA during transcription. This interaction involves, in part, the base pairing of the tRNA accepter end with four bases in the bulge region of the T box antiterminator element, which prevents the formation of an alternative terminator element and results in complete transcription of the gene. In an effort to investigate the potential for molecular modulation of this mechanism, we developed a fluorescence anisotropy screening essay to identify ligands that can disrupt the tRNA-T Box antiterminator interactions in a model system. Two sets of small molecules were investigated and the interaction between the lead compounds and T Box antiterminator was studied in more details using Fluorescence Resonance Energy Transfer (FRET) and enzymatic probing.
Keywords: T box transcription antitermination, Fluorescence anisotropy, Small molecule inhibitors
Abstract:
T box antiterminator riboswitch regulation is found primarily in Gram-positive bacteria and is a major regulatory mechanism for genes related to amino acid biosynthesis. The 5’ untranslated region of these genes monitor and respond to the charging ratio of cognate tRNA. The ultimate objective of this project is to develop novel RNA-targeted medicinal agents through examination of the structure and function of the RNA and associated ligands. A more specific aim is to find peptides that are capable of binding to the antiterminator element of the Bacillus subtilis tyrS sequence to modulate the tRNA-antiterminator complex critical for the functioning of the T box riboswitch. The antiterminator contains the highly conserved T box sequence and a seven nucleotide bulge, of which the first four nucleotides base pair with the tRNA in the complex. The model antiterminator, AM1A, contains the same T box sequence and seven nucleotide bulge and is used in binding studies with various peptide sequences. The goal is to find peptides that will bind well with the antiterminator and then place the ligand complex in further testing to identify the effect of the binding on the tRNA-antiterminator interaction. Fluorescence spectroscopy was used to determine two trends: first, the relative binding affinities of a small library of peptides to the AM1A and second, the effect of the relative position of the amino acids within the peptide on the relative binding affinity. Optimization of the assay method will be presented.
Keywords: T box, peptide
