RRM
2006
Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Gene prediction in Dydimium iridis is complicated because of frequent shifts in the reading frame due to mRNA editing. Here, we apply our algorithm Predictor of Insertional Editing (PIE) that has previously been tested on the related organism Physarum polycephalum. We choose the nad6 gene as our test case. We build a position specific scoring matrix (PSSM) which summarizes the multiple alignment of all known nad6 protein sequences in other organisms. Then we align this PSSM to the mitochondrial genome of Dydimium mitochondria. We identified the general location of the nad6 gene. The positions of the start and stop codon were determined by a statistical analysis of predictions from 111 PSSMs. The predicted mRNA sequence for nad6 has a length of 466 bases and 11 editing sites. While an experimental verification of our prediction is still outstanding, the high statistical significance of the prediction indicates that PIE is applicable to Didymium sequences and that PIE is likely able to predict other genes in Dydimium. This work will thus help discover edited genes in the mitochondrial genome of Didymium iridis and provide some clues about the mechanism of mRNA editing.
Keywords: RNA editing, computational prediction
Abstract:
Many biological processes, including gene regulation, binding of transcription factors, and DNA packaging, require the formation of DNA loops. We posit that melted segments of double-stranded DNA (dsDNA) are more flexible that closed dsDNA, and therefore that dsDNA constrained to a loop may melt locally to reduce the overall bending energy for the loop. We computationally predict sites where local melting may be observed in cyclized dsDNA molecules approximately 100 basepairs (bps) in length. The free energy cost of melting a segment of dsDNA is taken to be the sum of the stacking energies for the open bps and the loop entropy for the open segment. We use a generalized semiflexible polymer model, where melted sites may have elasticities that differ from that of closed dsDNA, and estimate the flexiblilty of these sites needed to explain results of cyclization experiments. The predictions presented here may be compared with experimental measurements of the frequency with which melting occurs made using Forster resonance energy transfer (FRET).
Keywords: DNA loops, DNA melting
Abstract:
Lysyl-tRNALys can be synthesized by a Lysyl-tRNA synthetase (LysRS) belonging to either synthetase class I (LysRS1), or class II (LysRS2). LysRS is the only synthetase known to violate the class rule, leading to questions as to why nature selected two different enzymes for performing the same function. The solving of the crystal structure of LysRS1 from Pyrococcus horikoshii gave some insight to this question. The structure solved with and without L-lysine revealed the composition of the lysine binding pocket. Comparison with the structure of LysRS2 from E. coli showed the LysRS1 binding pocket to be more compact than the one for LysRS2. In vitro analysis with L-lysine analogues supported this assertion as the Kis for S-(2-aminoethyl)-L-cysteine and L-lysinamide were at least 180 fold lower for LysRS2 than LysRS1. In contrast, gamma-aminobutyric acid was found to have a much lower Ki for LysRS1 than LysRS2. These differences were confirmed through in vivo experiments.
The LysRS1 crystal structure revealed aspects of the way tRNA binds with the enzyme. The anticodon loop of the tRNA is recognized by an alpha-helix cage domain that is only found in one other synthetase. While the structure could not be solved with tRNA bound to the enzyme, modeling was done to determine how the enzyme might recognize the tRNA. From this model amino acids that bind the anticodon nucleotides on tRNALys were identified. Variant enzymes were then created along with tRNALys mutants that contain different anticodon nucleotides. Kinetic analysis was done by finding the kcat/KM for each enzyme-tRNA combination. The intent of this project is to combine the biochemical data with energy minimization modeling to build a new docking mock-up. Discovering how LysRS1 binds the anticodon loop of its tRNA could provide interesting contrast when compared to the better studied OB-fold domain of LysRS2.
References:
Jester, B., Levengood, J., Roy, H., Ibba, M., and DeVine, K. (2003) Proc. Natl. Acad. Sci. USA 10014351-14356.
Levengood, J., Ataide, S., Roy, H., and Ibba, M. (2004) J. Biol. Chem. 279:17707-17714.
Terada, T., Nureki, O., Ishitani, R., Ambrogelly, A., Ibba, M., Söll, D., and Yokoyama, S. (2002) Nat. Struct. Biol. 9(4):257-262.
Keywords: synthetase, tRNA
Abstract:
PTC-containing human beta-globin mRNA is commonly used to study NMD in non-erythroid cells, where it decays in association with the nuclear fraction. In erythroid cells a PTC at codon 60/61 activates cytoplasmic endonuclease decay but has no impact on steady state mRNA in the nuclear fraction. We sought to determine the relationship between the cytoplasmic endonuclease-mediated decay of beta-globin mRNA in erythroid cells versus non-erythroid cells, and asked if the location of the PTC determines the sites of endonuclease cleavage. Beta-globin genes were generated with PTCs at codons 17, 22, 35, 39, 43, 60/61 and 90, and these were transiently transfected into murine erythroleukemia (MEL) cells. Steady-state levels of beta-globin mRNA were quantified in cytoplasmic and nuclear extracts by real-time PCR, and sites of endonuclease cleavage by S1 nuclease protection assay. In agreement with earlier work PTC-containing beta-globin mRNA is selectively lost from the cytoplasmic fraction of these cells, with the greatest impact on mRNAs with PTCs at codons 35, 39, 43, and 60/61. This was reversed by transfecting cells with dominant negative forms of Upf1, indicating cytoplasmic endonuclease decay constitutes a form of surveillance for beta-globin mRNA in erythroid cells. In addition we find that the location of the PTC has no impact on the position of endonuclease cleavage sites. When the same genes were analyzed in Cos-1 cells PTC-containing beta-globin mRNA is lost from the nuclear fraction, and with no evidence for production of endonuclease decay intermediates. These results demonstrate that different mechanisms are involved in the decay of PTC-containing beta-globin mRNA in erythroid versus non-erythroid cells.
Keywords: Nonsense-mediated decay, beta-globin, endonuclease
Abstract:
Pseudouridine synthases (Ψ synthases) catalyze the isomerization of uridine to pseudouridine (Ψ) and are present in all domains of life. The Ψ synthases fall into five families, and crystal structure of members of each family show that they share the same core fold. The cocrystal structure of the E. coli Ψ synthase RluA bound to an RNA oligomer corresponding to the anticodon stem-loop (ASL) of E. coli tRNAPhe has recently been determined by us (Hoang, C., et al., and Ferre-D'Amare, Mol. Cell, in press). This structure is only the second of a Ψ synthase bound to an RNA substrate. In the RluA-RNA cocrystal, the RNA has undergone significant conformational changes from its unbound form due to protein-RNA interactions. To test the importance of particular interactions in the RluA-ASL cocrystal structure, the enzymatic activity of RluA towards seven altered ASLs was measured. These kinetic studies are presented along with the active site structure.
Keywords: pseudouridine synthase, RluA, RNA recognition
Abstract:
Exosome complexes are composed of 3’ to 5’ exoribonucleases that are essential for viability, conserved in eukaryotes and archaea, and critical for numerous distinct RNA metabolic pathways. Previous studies have implicated specific exosome subunits in chromosome segregation and cell division, but the exact mechanisms underlying these functions have remained unclear or considered indirect. We find that endogenous exosome subunits dynamically and differentially redistribute during mitosis and cytokinesis in Drosophila melanogaster S2 tissue culture cells. For example, dRrp6 is recruited to centrosomes during prophase and prometaphase, then accumulates on the perichromosomal layer during metaphase, anaphase, and telophase, and finally is targeted to the midbody and partitioned centrosomes during cytokinesis. In contrast, dRrp4, dRrp40, and dRrp46 are almost exclusively, yet differentially, enriched on microtubular structures including the spindle, astral microtubules, spindle remnant, and midbody. Treatment of S2 cells with drugs that perturb microtubule architecture elicits subunit- and stage-specific localization patterns. RNA interference of certain exosome subunits leads to a decrease in mitotic cells with an increase in aberrant chromosome segregation events. These results suggest that exosome subunits have discrete interactions and functions during distinct stages of cell division and that the signaling pathways regulating subunit mobilization and interplay are complex and tightly regulated.
We further explored exosome subunit function in cell division in the budding yeast, Saccharomyces cerevisiae. Notably, several yeast exosome subunit mutants are defective in mitotic progression as evidenced by an increase in cells harboring pre-anaphase spindles. These spindles are frequently misoriented along the mother-bud axis and mispositioned away from the bud neck. Moreover, exosome subunit mutant cells contain elongated astral microtubules. Finally, consistent with a role for exosome subunits in regulating microtubule structure, exosome mutant cell growth is inhibited by microtubule destabilizing agents. Given that RNA is required for spindle assembly in Xenopus (Blower et al., 2005), we hypothesize that exosome subunits directly participate in a conserved microtubule-based mechanism that is required for proper mitotic progression.
References:
M. Blower, M. Nachury, R. Heald, K. Weis. (2005) A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly. Cell. 121(2); 223-234.
Keywords: exosome, mitosis, cytokinesis
Abstract:
Although RNA editing is widespread in nature, cytidine (C) to Uridine (U) editing of tRNA has only been found in organelles (mitochondria and chloroplast). We show here that all three tRNAThr isoacceptors in Leishmania tarentolae and Trypanosoma brucei undergo C to U editing in the nucleus. This represents the only known tRNA C to U editing event that occurs outside of mitochondria or chloroplasts. The significance of this observation rests in the fact that when looking at editing levels, for each isoacceptor, aminoacylated tRNAs are edited to a higher extent than what is found in the total tRNA population. Also, in all cases, a higher percentage of the tRNA is edited in cytoplasmic as compared to nuclear RNA fractions. Taken together these observations imply that the edited species are not only functional in these organisms but while produced in the nucleus, they are preferentially exported to and aminoacylated in the cytoplasm. These findings also indicate that tRNA editing is more widespread than previously thought and it is not limited to eukaryotic organelles.
Keywords: tRNA, editing, kinetoplastid
Abstract:
Bacillus subtilis tyrS is an aminoacyl-tRNA synthetase gene that is regulated by the T box transcription antitermination mechanism. Expression of this gene is regulated by the interaction of uncharged tRNATyr with the 5' untranslated region of the nascent mRNA. One feature of this interaction is the base pairing of the tRNA acceptor end with four bases of a highly conserved mRNA structural element termed the antiterminator. This tRNA-antiterminator base pairing prevents the formation of an alternative terminator element and results in antitermination of transcription. The binding of small molecules to the antiterminator model RNAs was investigated to determine ligand structure-activity relationship for binding and to investigate the effect of antiterminator sequence differences on binding specificity. AM1A and AM1A(C11U) were studied using fluorescence resonance energy transfer (FRET). AM1A(C11U) is a reduced function variant of the wild-type model AM1A and has subtle but significant structural differences compared to AM1A. Oxazolidinones and aminoglycosides were the classes of RNA ligands investigated. Several ligands were identified which bond selectively to AM1A. Ligands with high affinity to AM1A were also investigated via enzymatic footprinting and molecular modeling to identify the site of binding.
Keywords: FRET, Footprinting, Oxazolidinones
Abstract:
Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing (occurring in all domains of life), cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNAThr(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine containing tRNAThr(AGU) is also edited at position 32. In vitro, C to U editing stimulates the essential conversion of A to I at the wobble base. Surprisingly, enzymes mediating tRNA deamination in bacteria and yeast contain conserved cytidine deaminase motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, the enzyme responsible for either reaction has not been identified. Here, we show that one trypanosomatid enzyme TbADAT2p can mediate A to I editing in vitro and is required for both editing reactions in vivo. Thus, a single deaminase can mediate two tRNA editing events, providing a model for a multi-specificity editing enzyme. We suggest that the observed flexibility of the TbADAT2 protein is an inherent characteristic of the ancestor deaminase domain. Substrate specificity of this enzyme family might have then evolved by appending this flexible catalytic module onto different protein scaffolds.
Keywords: tRNA, Editing, Deamination
Abstract:
Since the discovery of a third tRNA binding site, the exit (E) site, on the ribosome, two models for its function have emerged(1-3). The first model posits that the E site interacts with the tRNA moving out of the P site and thereby contributes to the mechanism of translocation(4). The second model suggests that E-site tRNA contributes to translational fidelity by stabilizing mRNA through codon-anticodon pairing until a cognate aminoacyl-tRNA is delivered to the A site, at which point the E-site tRNA rapidly dissociates(2). Recent crystal structures predict hydrogen bonding between the 3'; terminal adenosine (A76) of tRNA and C2394 of the 50S E site, and both of these nucleotides are universally conserved(5-7). In previous functional studies, a number of modifications of A76 of P-site tRNA decreased the rate of translocation, and these decreased rates correlated well with decreased E-site binding affinities(4). Here we approach the question of E-site function by mutating C2394 in a strain of E.coli that contains a single copy of the 23S gene. Ribosomes with substitutions at position 2394 are able to support growth with an ~2-fold slower growth rate, but these ribosomes are inactive in an in vitro translation assay. We also show that these substitutions decrease the rate of translocation in vitro by up to 80-fold and provide independent evidence that these mutations inhibit P/E state formation. From this we conclude that rapid translocation is dependent on C2394 of the 50S E site, consistent with a role for this site in the translocation mechanism. At the same time, the fact that these mutations confer unexpectedly mild growth phenotypes suggests that their defects are somehow compensated for in vivo.
References:
1. Rheinberger, H.J., Sternbach, H. and Nierhaus, K.H. (1981) Proc Natl Acad Sci U S A, 78, 5310-5314.
2. Rheinberger, H.J. and Nierhaus, K.H. (1983) Proc Natl Acad Sci U S A, 80, 4213-4217.
3. Robertson, J.M., Urbanke, C., Chinali, G., Wintermeyer, W. and Parmeggiani, A. (1986) J Mol Biol, 189, 653-662.
4. Lill, R., Robertson, J.M. and Wintermeyer, W. (1989) Embo J, 8, 3933-3938.
5. Korostelev, A., Trakhanov, S., Laurberg, M. and Noller, H.F. (2006) Cell.
6. Selmer, M., Dunham, C.M., Murphy Iv, F.V., Weixlbaumer, A., Petry, S., Kelley, A.C., Weir, J.R. and Ramakrishnan, V. (2006) Science.
7. Schmeing, T.M., Moore, P.B. and Steitz, T.A. (2003) RNA, 9, 1345-1352.
Keywords: ribosome, E site, translocation
Abstract:
Dihydrouridine is a modified nucleoside present in the tRNAs of eubacteria, eukaryotes, and some archea. This modified nucleoside is formed by the reduction of the double bond in uridine at specific sites in tRNAs, most commonly the D-loop region. The family of proteins catalyzing this reaction, dihydrouridine synthases, have only recently been identified in E.coli and S. cerivesiae. We have cloned, expressed, and partially characterized a member of this family of proteins from the thermophilic bacterium Thermotoga maritima. This protein is a 38kD monomeric protein which utilizes an FMN prosthetic group. Catalysis is comprised of a reductive half reaction, in which NADPH reduces the flavin, and an oxidative half-reaction whereby the flavin reduces the double bond of uridine. The reductive half-reaction at 25 degrees Celcius shows an apparent limiting rate constant of 2.3 x 10-4 s-1 with a KD of 600 μM. Stereochemistry of the hydride trandsfer shows the enzyme prefers the pro-R face of NADPH, however no kinetic isotope effect is seen for this reaction, indicating that chemistry is not rate limiting. Oxidation of the enzyme by a non-native pre-tRNAtyr reoxidizes the flavin with a rate of 3.2 x 10-5 s-1.
Keywords: dihydrouridine, thermotoga, kinetics
Abstract:
Metagenomics has been employed to systematically sequence, classify, analyze, and manipulate the entire genetic material isolated from environmental samples. Finding genes within metagenomic sequences remains a formidable challenge and noncoding RNA genes other than those encoding rRNA and tRNA are not well annotated in metagenomic projects. In this work, we identify, validate, and analyze the genes coding for RNase P RNA (P RNA) from all published metagenomic projects. P RNA is the RNA subunit of a ubiquitous endoribonuclease RNase P that consists of one RNA subunit and one or more protein subunits. The bacterial P RNAs are classified into two types, Type A and Type B, based on the constituents of the structure involved in precursor tRNA binding. Bacterial and some archaeal P RNAs are catalytically active without protein subunits, capable of cleaving precursor tRNA transcripts to produce their mature 5΄-termini. We have found 328 distinctive P RNAs (320 bacterial and 8 archaeal) from all published metagenomics sequences, which led us to expand by 60% the total number of this catalytic RNA from prokaryotes. Surprisingly, all newly-identified P RNAs from metagenomics sequences are of Type A. One of the distinctive features of some new P RNAs is that the P2 stem has kinked nucleotides in its 5’ strand. We find that the single nucleotide J2/3 joint region linking the P2 and P3 stem that was used to distinguish a bacterial P RNA from an archaeal one is no longer applicable, i.e. some archaeal P RNAs have only one nucleotide in the J2/3 joint. We also discuss the phylogenetic analysis based on covariance model of P RNA that offers a few advantages over the one based on 16S rRNA.
Keywords: rnase p rna, metagenomics
Abstract:
CUG binding protein 1 (CUG-BP1) and muscleblind-like protein 1 (MBNL1) have been shown to bind to discrete sites in several of the same pre-mRNAs and regulate cell-specific alternative splicing in an antagonistic manner. We previously proposed that a change in the balance between CUG-BP1 and MBNL1 activities in the developing heart drives a fetal-to-adult transition in cardiac troponin T (cTNT) alternative splicing. To investigate whether CUG-BP1 and MBNL1 also developmentally regulate alternative splicing during embryonic cardiogenesis, we examined CUG-BP1 and MBNL1 expression in the developing chick. Chicken CUG-BP1 and MBNL1 are 94% and 96% identical, respectively, to their human orthologs at the amino acid level. CUG-BP1 is first detected in the developing heart by in situ hybridization during fusion of the primitive heart tube (H&H st.8-10), concomitant with the onset of cardiac differentiation. Expression continues in the heart during cardiac morphogenesis, as the heart tube undergoes looping (st.10-14), the myocardium undergoes trabeculation and endocardial cushion outgrowth septates the heart into chambers (st.18-23). In contrast, MBNL1 is not detected until looping is well underway and induction of endocardial cushion outgrowth has begun (st.14). Western blot analyses revealed that although CUG-BP1 is continuously expressed in the embryonic heart (st.10-35), there is a strong, transient up-regulation of CUG-BP1 protein during later stages of cardiac morphogenesis (st.18-29). During this same time, MBNL1 undergoes a dramatic isoform transition without an appreciable change in the overall level of expression. These changes in CUG-BP1 and MBNL1 expression coincide with a change in the pattern of cTNT alternative splicing, a known pre-mRNA target, as well as several other alternative splicing transitions in the heart. Together these results suggest that CUG-BP1 and MBNL1 proteins not only mediate fetal-to-adult changes in splicing, but also influence developmentally regulated splicing events during embryonic cardiac morphogenesis.
Keywords: alternative splicing, CUG-BP1, MBNL1
Abstract:
The T box regulatory system in Gram-positive bacteria utilizes a complex set of conserved structural and sequence elements in the 5' untranslated region of the transcript to regulate the expression of a large number of amino-acid related genes through premature termination of transcription. Expression of each gene regulated by this mechanism is induced by a reduction in the aminoacylation level of the cognate tRNA. A purified in vitro transcription system and structural mapping studies have directly demonstrated that binding of the uncharged cognate tRNAGly to the leader region of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) stabilizes an antiterminator element that competes with formation of the intrinsic terminator helix. T box RNAs are therefore members of the riboswitch family, since the RNA rearrangement occurs in response to direct binding of the effector molecule. The specificity of the leader RNA-tRNA interaction is determined primarily by base pairing between a single codon in the leader RNA and the tRNA anticodon, and between the antiterminator and the unpaired residues at the acceptor end of the tRNA. However, additional interactions are likely to be required for efficient binding, and little is known about the tertiary structure of the leader RNA and the arrangement of leader RNA domains relative to the tRNA. In this study, a set of variants with alterations in the glyQS glycine codon and corresponding variants with alterations in the tRNAGly anticodon were tested to determine the effect of specific codon-anticodon mismatches on antitermination activity.
Keywords: T box, riboswitch, transcription termination
Abstract:
Polyadenylation in plants requires at least three cis elements. They are far upstream element (FUE), near upstream element (NUE) and cleavage/ polyadenylation site. These elements act as poly(A) signals and are recognized by a set of polyadenylation factors. Cleavage stimulation factor (CstF) is one of the factors with three different subunits of 77, 64 and 50 kD. The CstF complex plays a main role in the recognition of the downstream GU rich element and aids in cleavage along with other cleavage factors. The present study focuses on the Arabidopsis ortholog of CstF50. Yeast two hybrid assays were employed to study the interactions of CstF50 with other polyadenylation factor subunits. The results of yeast two hybrid shows the positive interactions with CPSF100, CPSF30, two different PAP isoforms, one PabN isoform, and Fip1. The CstF50 interaction with poly(A) polymerase and poly(A) binding protein suggests that it is involved both in cleavage and polyadenylation. These results indicate that plant CstF50 is different from the mammalian CstF50 which has only the cleavage function. Further sub-cellular localization studies are being conducted by fusing CtstF50 with DsRed. Progress along these lines will be presented.
References:
1. Li, Q. and A. G. Hunt (1997). The Polyadenylation of RNA in Plants. 115: 321-325.
2. Takagaki, Y., J. L. Manley, et al. (1990). A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. 4: 2112-2120.
Keywords: Polyadenylation, Yeast two hybrid assay, CstF
Abstract:
A critical interaction that occurs during the T box transcriptional antitermination regulatory mechanism involves a unique RNA-RNA interaction. The acceptor end of an uncharged cognate tRNA interacts with 5'-end nucleotides in a 7 - nucleotide bulge of an otherwise unstable antiterminator element to stabilize it. The nucleotides that form the bulge of the antiterminator are the middle 7 sequence of the 14-nucleotide T box sequence, which is highly conserved. The significance of the conservation of the 3'-end nucleotides of the bulge and in the A2 helix above the bulge, though unknown, has been found to be very important for function. To determine whether the antiterminator and tRNA requirements for functional antitermination are determined solely at the level of RNA-RNA interactions, a series of antiterminator and tRNA model RNA have been investigated. The antiterminator RNAs were selected based on phylogenetic-derived sequence changes and bulge sequences derived from in vitro evolution studies. Affinities and general structural characteristics between the antiterminator models and tRNA analogs were investigated using fluorescence anisotropy, enzymatic probing and circular dichroism. Our results show that there are structural differences between the antiterminator RNAs and that they bind the tRNA analogs with differing affinities. Although there were some exceptions, our results correlate very well with in vivo and in vitro antitermination efficiency data. The implications of the findings from this study will be discussed in detail.
Keywords: Antiterminator, Phylogenetic, Anisotropy
Abstract:
A unique RNA/RNA interaction occurs between uncharged tRNA and the mRNA 5' leader region of many Gram-positive bacterial tRNA synthetase, amino acid biosynthesis, and amino acid transport genes (1). This interaction leads to antitermination of transcription and complete transcription of the gene. Without this interaction (i.e. in the presence of only charged tRNA) transcription termination occurs (2). The current investigations revolve around the structural features involved in the recognition of the tRNA acceptor stem by the antiterminator, containing a unique seven-nucleotide bulge, in the leader region (3,4). Fluorescence techniques using 2-aminopurine as well as NMR spectroscopy have been used to probe structural changes in the antiterminator upon binding with a microhelix tRNA model.
References:
1. Grundy, F.J.; Moir, T.R.; Haldeman, M.T.; Henkin, T.M. (2002) Nucleic Acids Res. 30, 1646-1655.
2. Grundy, F.J.; Collins, J.A.; Rollins, S.M.; and Henkin, T.M. (2000) RNA 6, 1131-1141.
3. Gerdeman, M.S.; Henkin, T.M.; Hines, J.V. (2002) Nucleic Acids Res. 30, 1065-1072.
4. Gerdeman, M.S.; Henkin, T.M.; Hines, J.V. (2003) J. Mol. Biol. 326, 189-201.
Keywords: mRNAtRNA interaction, fluorescence, NMR
Abstract:
Mpp10p is a target of autoantibodies produced in the human autoimmune disease systemic sclerosis. This essential protein is part of the small subunit (SSU) processome that initiates SSU biogenesis by releasing the 20S SSU precursor from the pre-rRNA via three endonucleolytic cleavages. In Saccharomyces cerevisiae, the model system for our studies, this processome contains approximately 39 other proteins and the U3 small nucleolar RNA (snoRNA). The role of most SSU processome proteins is unclear. Our studies aim to decipher the role of three processome proteins: Mpp10p and two of its interacting partners Imp3p and Imp4p. Previously, we determined that Imp3p and Imp4p mediate annealing between two specific sites of the U3 snoRNA and two complementary sites of the pre-rRNA (Gerczei and Correll (2004) PNAS 101, 15301). The annealing activities of Imp3p and Imp4p permit rapid and complete U3-pre-rRNA hybridization by removing kinetic and thermodynamic barriers. Here, we present the first biochemical evidence of direct interaction between Mpp10p and the U3 snoRNA. Foot printing studies indicate that Mpp10p binds specifically to the U3 snoRNA (Kd ~200 nM). The regions protected by Mpp10p include nucleotides in box B, box C¡¯ and the hinge region previously identified as essential for the association of Mpp10p with the SSU processome (Wormsley et al (2001) RNA 7, 904). Experiments are underway to determine how the presence of Mpp10p affects the U3-pre-rRNA annealing activities of Imp3p and Imp4p and to determine whether Mpp10p possesses inherent annealing activity.
Keywords: Ribosome biogenesis, Mpp10p, SSU processome
Abstract:
The E. coli aroL gene, encoding shikimate kinase II, contains an untranslated leader of 124 nucleotides (nts) that includes a canonical Shine-Dalgarno (SD) sequence positioned appropriately upstream to the aroL start codon. Ribosome binding studies (i.e., toeprint assays) revealed 30S ribosomal subunits bind at the aroL start codon and to one of two, or both, AUG triplet(s) located within the untranslated leader, upstream to the aroL start codon. Neither upstream start codon contains a SD sequence. AUG (number 1) is at position –67 relative to the aroL start codon and contains an in-frame stop codon nine nts away. The open reading frame specified by AUG (number 2), at position –62, encodes a putative peptide of 21 amino acids. The stop codon (UGA) in frame with AUG (number 2) overlaps the aroL start codon, suggesting aroL could be translationally coupled to the upstream AUG (number 2) open reading frame. Translational fusion of the AUG (number 1) open reading frame to a lacZ reporter gene suggests that it has minimal activity for initiating translation. Fusion of the AUG (number 2) reading frame to lacZ reveals a significant amount of translational activity, producing approximately 30 percent of the LacZ activity measured when the aroL coding sequence is fused to lacZ. Site-directed mutation of the AUG (number 1) had negligible effect on translation from the downstream aroL start codon whereas mutation of AUG (number 2) reduced expression from aroL by approximately 30 percent. Identification of the signals used by ribosomes to bind AUG (number 2), in the absence of a SD sequence, might reveal novel signals used by ribosomes to find and bind translational start sites in other mRNAs.
Keywords: Translational Coupling, Ribosome Binding, untranslated leader
Abstract:
During translation initiation in bacteria, the start codon is recognized by the initiator tRNA (fMet-tRNAfMet) in the 30S P site. The initiation factors (IF1, IF2, and IF3) are thought to work in conjunction with the 30S subunit to ensure faithful recognition of the start codon. Mutations in infC, the gene encoding IF3, cause spurious initiation at non-canonical start codons, suggesting an important role for IF3 in initiation fidelity. Here, we characterize two 16S rRNA mutations, G1338A and A790G, which decrease initiation fidelity in vivo. G1338 is a universally conserved nucleotide that interacts with the minor groove of the anticodon stem of P-site tRNA, forming a type II interaction. Previous genetic studies have shown that G1338A can suppress other P-site mutations, suggesting that G1338A may increase the affinity of tRNA for the 30S P site. Indeed, 30S subunits harboring G1338A have increased affinity for tRNAfMet in vitro, suggesting that the fidelity defect may result from a more favorable type II A minor interaction with the initiator tRNA. When G1338A is combined with various infC alleles, effects of the rRNA and protein mutations are consistently additive, suggesting that G1338 and IF3 may affect fidelity independently. Mutation A790G seems to act in a different way to decrease fidelity. A790G decreases the affinity of IF3 for the 30S subunit substantially, and does not confer an increased fidelity defect when combined with infC mutations. These data suggest that A790G may decrease fidelity by inhibiting IF3 interaction with the 30S subunit.
References:
Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli. Abdi NM, Fredrick K RNA 2005 Nov 11 (11) 1624-32.
Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V. Nature 2000 Sep 21;407 (6802):340-8
Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo. Sussman JK, Simons EL, Simons RW. Mol Microbiol.1996 Jul; 21(2):347-60.
Keywords: G1338, A790, if3
Abstract:
Many Gram-positive bacterial genes use the T box transcription antitermination mechanism to control transcription via a RNA-RNA interaction between uncharged, cognate tRNA and the mRNA leader region of the gene transcript. The tRNA is recognized by at least two regions of the leader. The specifier sequence, or codon, binds the anticodon of the tRNA, and the antiterminator binds the acceptor end of the uncharged tRNA. Previous studies of this system have shown that the secondary structure of the mRNA leader region, tertiary structure of the antiterminator, and the complex with the uncharged tRNA play a fundamental role in antitermination in vivo and tRNA binding in vitro. Interest in the stability and versatility of RNA structure formation has identified Mg2+ as a critical factor in the folding, structural integrity, and biological function of RNA. The hypothesis of this research is that Mg2+ is involved in direct interactions with the T box antiterminator as well as in the stabilization of the mRNA/ tRNA binding event. These studies are important in order to understand the structure-function relationship of this novel regulatory system. Spectroscopic (UV, CD, NMR) as well as terbium (III) footprinting data representing the effects of Mg2+ and its relevant probes [Co(NH3)63+ and Mn2+ on the antiterminator and its complex with model tRNA will be presented.
Keywords: mRNA-tRNA interaction, metal ions, NMR
Abstract:
Internal motions in RNA can be decoupled from overall reorientation by elongating a given reference domain using unlabelled Watson-Crick residues. Here, we show that by elongating more than one domain it is possible to collect larger amounts of NMR data, including spin relaxation measurements and residual dipolar couplings, which provides a view of dynamics relative to multiple frames of reference. We show that this “inverse” domain elongation approach resurrects sensitivity to twisting motions around axially symmetric domains allowing model-free characterization of motions with complete rotational sensitivity. The multi-domain elongation approach is demonstrated on the HIV-1 Transactivation Response Element (TAR). Results reveal a combination of bending and twisting domain motions that mirror the structural changes that follow adaptive recognition of diverse chemical targets. Implications of results on the rational design of TAR binding anti-HIV therapeutics will also be discussed.
Keywords: residual dipolar couplings, HIV-1 TAR, dynamics
Abstract:
Resolving and characterizing internal motions based on NMR spin relaxation and residual dipolar couplings (RDCs) methods requires that internal motions are not correlated to overall reorientation. Here, we describe implementation of an elongation strategy which allowed us to decouple internal motions in ribonucleic acids (RNA) from overall reorientation. Application of spin relaxation and RDC methods revealed hierarchical motional modes spanning picosecond librations, nanosecond collective motions of A-form helical domains, and micro-to-millisecond structural transitions. Our results uncover a universe of internal motions occurring at nanosecond timescales that evade detection by conventional NMR spin relaxation methods because they occur at timescales approaching overall rotational diffusion. We present direct evidence that this uncovered network of motional modes code for functionally important changes in RNA conformation that allow adaptive recognition of chemically diverse targets.
The domain-elongation strategy provides a new approach for resolving motional modes in RNA spanning picosecond to millisecond timescales. Our results suggest that the adaptive changes in RNA conformation that take place upon ligand binding actually occur dynamically in the free RNA. Thus a manifold of internal motional modes codes for RNA adaptation.
Keywords: RNA dynamics, adaptive recognition, NMR
Abstract:
The transactivation response element (TAR) RNA has multiple roles in HIV replication, including transcriptional elongation. Once bound to TAR, the viral protein Tat recruits the transcription elongation factor, P-TEFb, which eventually leads to phosphorylation of the RNA polymerase and thus transcriptional elongation.(1) Both an internal bulge and apical loop in TAR are believed to be essential for proper binding of Tat and P-TEFb. To date, NMR studies that have investigated the structural dynamics of TAR have used a TAR construct in which the six-residue hairpin loop was replaced with a UUCG tetraloop. Here, we report initial steps towards complete NMR characterization of the structural dynamics of wt-TAR and its Tat binding properties. Our results indicate that mutation of the apical loop has minimal effects on the stem-bulge-stem element of TAR. This is in contrast to a previous biochemical study indicating formation of a U31•A22-U40 base-pair.(2) The wild-type loop undergoes complex internal motions spanning nanosecond to millisecond timescales. The NMR data will be compared with a 65 ns molecular dynamics simulation of wt-TAR in explicit solvent.
References:
1 Price, D. H. (2000). P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 20, 2629-34.
2 Huthoff, H., Girard, F., Wijmenga, S. S. & Berkhout, B. (2004). Evidence for a base triple in the free
Keywords: TAR, NMR, dynamics
Abstract:
Bacterial manganese superoxide dismutase (MnSOD) has been found to associate with double stranded DNA based on in situ immunostaining, gel retardation and nitrocellulose assays. Since then, no studies have been published with regard to MnSOD-RNA interactions. Also, no systematic variation of solution conditions, or thermodynamics, have been reported. We present evidence of fluorescence quenching upon binding single-stranded (ss) RNAs (poly(U), poly(C) and poly(A)) as well as double stranded plasmid DNA. We report the binding affinity (Kobs) as a function of monovalent salt concentration for each. Additionally, we have measured Kobs at different temperatures, leading to van’t Hoff enthalpy determinations, also as a function of [salt]. Surprisingly, at any given [salt], the binding affinity of MnSOD for ss RNAs is higher than for dsDNA, indicating that although MnSOD has the potential to bind chromosomal DNA, its main function may be to bind either single-stranded regions of DNA (e.g., at replication forks or transcription bubbles) or to mRNAs.
Keywords: Manganese Superoxide Dismutase, single-stranded RNA, fluorescence
Abstract:
We used a domain elongation strategy and Nuclear Magnetic Resonance (NMR) spin relaxation and residual dipolar coupling methods to examine how shortening the length of the TAR RNA bulge linker from three (HIV-1 form) to two (HIV-2 form) residues affects its structural dynamics and corresponding ability to adaptively bind diverse targets. We observe up to ~65% decrease in the amplitude of inter-domain motions occurring at both nanosecond and micro-millisecond timescales in HIV-2 TAR as compared to HIV-1 TAR. The reduced domain motional amplitudes originate from decreased local flexibility occurring at nanosecond timescales at the backbone of bulge and neighboring stem residues. In contrast, increased flexibility is observed in the nucleobases of HIV-2 TAR which can be explained by looping out of the bulge residues owing to greater coaxial stacking of the two domains. Analysis of RDCs shows that while HIV-1 TAR can be modeled as inter-converting among its previously reported bound structures, only a subset of the bound conformers are needed to re-construct the corresponding dynamical ensemble of HIV-2 TAR. Remarkably, the HIV-2 TAR conformers are precisely those that have the most linear inter-domain alignments. These results indicate that HIV-2 TAR is less predisposed to adaptively form bent conformations that can be formed by HIV-1 TAR. Binding data in support of this hypothesis will be presented.
Keywords: RNA dynamics, adaptive recognition, NMR
Abstract:
Chemical shift perturbations occupy an important facet in the study of biological molecules by nuclear magnetic resonance (NMR) spectroscopy. Given the complexity of their interpretation, however, they are often used as a rudimentary means to measure dissociation constants or to determine the relative binding site of a drug. Residual dipolar couplings (RDCs) on the other hand represent a sensitive technique which is un-obscured by the complexity that plagues chemical shifts and demonstrates a particularly useful method by which one may acquire structural data. By combining the chemically sensitivity nature of chemical shift perturbations with the structural sensitivity of RDCs we have been able to more completely characterize binding modes of aminoglycosides to RNA. In particular, the strategy yields a description of RNA binding sites and RNA structural changes that follow recognition. Especially when considering the malleability of RNA and the structural flexibility of the drugs they encounter, such analyses of conformational adaptation is key for successful interpretation of structure-activity relationships. Using this approach, we have uncovered an unprecedented degree of structural adaptability accompanying the recognition of HIV-1 TAR RNA by structurally related aminolgycosides. We show that even the chemically similar Neomycin B (NeoB), Ribostamycin (Rib), and Kanamycin B (KanB) bind to very different conformational manifestations of the TAR RNA target. Our results suggest that even minimal structural differences can result in significant global and local changes to the RNA
Keywords: TAR, Aminoglycosides, Residual Dipolar Couplings
Abstract:
Mitochondria, though containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. Mitochondrial import of transfer ribonucleic acids (tRNAs) from the cytosol, if not universal, is widely spread among species, ranging from yeast to trypanosomatids and to plants. However, the identification of numerous specific tRNAs simultaneously in a complex mixture is challenging due to their similar physio-chemical properties. We present a ribonuclease-mediated cleavage coupled with MALDI-MS for the detection of cytosolic and mitochondrial tRNAs from Saccharomyces cerevisiae by their signature digestion products, which eventually pave the way to detect specific imported tRNAs in mitochondria.
A comparison of an organism’s complete complement of tRNA endonuclease digestion products yields a set of unique or “signature” digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. The mass spectrometric detection of any one or more of these signature products by multiple ribonucleases (RNase T1 and RNase A) from a complex mixture will confirm the presence of the cognate tRNA. We have already established and reported a method to detect specific prokaryotic tRNAs in a complex cellular ensemble by means of this signature digestion approach.
Transfer RNA detection was initially optimized using commercially available yeast tRNA mixtures. S. cerevisiae (YPH499) was grown in YEPD medium to an OD of 1.5-2.0. tRNA fractionation was optimized using hot phenol and SDS coupled with chloroform followed by digestion with ribonucleases. The digested mixtures were analyzed by MALDI-TOF MS and matched with their theoretical signature digestion products. Research is underway to separate and detect solely mitochondrial tRNAs from this organism. Once established, this methodology could be used to detect imported RNAs in other eukaryotes including human mitochondria.
Keywords: signature digestion products, tRNA import, Saccharomyces cerevisiae
Abstract:
We examined how static and dynamic deviations from the idealized A-form helix propagate into errors in the principal order tensor parameters determined using residual dipolar couplings (rdcs). A 20 ns molecular dynamics (MD) simulation of the HIV-1 transactivation response element (TAR) RNA together with a survey of spin relaxation studies of RNA dynamics reveals that pico-to-nanosecond local motions in non-terminal Watson-Crick base-pairs will uniformly attenuate base and sugar one bond rdcs by ~7%. Gaussian distributions were generated for base and sugar torsion angles through statistical comparison of 40 RNA X-ray structures solved to < 3.0 Å resolution. For a typical number of rdcs (≥ 11), these structural deviations together with rdc uncertainty (1.5 Hz) lead to average errors in the magnitude and orientation of the principal axis of order that are < 9% and < 4º respectively. The errors decrease to < 5% and < 4º for ≥ 17 rdcs. A protocol that allows for estimation of error in A-form order tensors due to both angular deviations and rdc uncertainty (Aform-RDC) is validated using theoretical simulations and used to analyze rdcs measured previously in TAR in the free state and bound to four distinct ligands. Results confirm earlier findings that the two TAR helices undergo large changes in both their mean relative orientation and dynamics upon binding to different targets.
Keywords: Alignment Tensor Error, Idealized A-form Helix, RDC
Abstract:
Polypyrimidine-tract-binding protein (PTB) is a highly conserved protein containing four RNA recognition motifs (RRM1-4), and tightly binds CU rich elements in precursor mRNAs. PTB plays an important role in the negative regulation of alternative splicing. Alternative splic-ing amplifies proteomic diversity from animal genomes. Misregulation of alternative splicing is frequently observed in human diseases involving the nervous system and cancer. However, the mechanisms that control alternative splicing regulation are complex and poorly understood. It has been proposed that PTB bridges the binding sites flanking a repressed exon, thus looping out the intervening RNA and sequestering it from the splicing machinery. Recent NMR structures of the individual RRMs support this hypothesis. [Oberstrass F.C. et al. (2005), Science 309:2054]
In order to further investigate the looping mechanism, we have prepared a series of ssRNA’s with the sequence 5’-CUCUCU(A)NCUCUCU-3’ (N = 5-30), and labeled their 5’ and 3’ ends with a fluorophore pair to monitor the conformational changes induced upon binding by PTB’s RRM3 and 4 using FRET. Our results clearly show that RRM3 and 4 binding brings the 5’ and 3’ ends of the ssRNA in close proximity supporting a mechanism where PTB loops out the intervening sequence. Furthermore, looping efficiency depends on the length of the interven-ing sequence, with optimal looping for loops 15-20 nucleotides long. Distance measurements using time resolved FRET yield donor-acceptor distances in agreement with the NMR structures. We are currently using single molecule FRET to further characterize the structural dynamics of the PTB-RNA complex and its role in alternative splicing regulation.
Keywords: PTB, Alternative Splicing, FRET
Abstract:
As in all reteroviruses, two identical copies of genomic RNA are packaged in the viral particles of the Human Immunodeficiency Virus Type I (HIV-1). The two RNA strands are held together in a non-covalent dimer the integrity of which is important in multiple steps of the viral life-cycle, including reverse transcription, recombination, RNA packaging, and viral infectivity. For this reason, SL-1 is a possible RNA target for developing anti-HIV therapeutics. In addition to a self-complementary palindromic loop, SL-1 contains a highly conserved G-rich bulge which has been shown to be critical for genome dimerization and packaging. Here, we show using NMR that the aminoglycoisde neomycin B binds specifically to the SL-1 bulge. The site of recognition maps to specific regions of high electronegative charge density where Mg2+ binding is also observed. We present initial data aimed at characterizing how neomycin B recognition affects the global and local structural dynamics of SL-1 and how this in turn compares with Mg2+ binding. When combined with previous studies showing neomycin B binding to the SL-1 loop, our data suggests that up to four unique binding sites in SL-1 dimers are available for targeting using therapeutic compounds.
Keywords: HIV-1 SL-1 RNA, NMR
Abstract:
A novel RNA-RNA interaction found in many gram-positive bacteria is an essential mechanism for the control of a unique class of genes called T box genes. This interaction leads to the formation and stabilization of an antiterminator structural element. Specifically, the 5’ untranslated leader region of the nascent mRNA transcript binds uncharged cognate tRNA. A 29-nucleotide antiterminator model has been designed with two flanking helices separated by a seven-nucleotide bulge. In order to study and understand this antiterminator-tRNA interaction in detail, the Circular dichroism (CD), fluorescence and fluorescence anisotropy techniques were employed to monitor what effects the pH has on antiterminator structure and binding of tRNA models to the antiterminator model RNA. Results and their implications regarding the structure-function of the antiterminator will be presented.
Keywords: antiterminator, Anisotropy, RNA-RNA interaction
Abstract:
Post-transcriptional modifications found in ribosomal RNA are thought to be important for both the structure and function of the ribosome. The modified nucleosides of rRNA tend to cluster in the functionally important region of the ribosome and modifications occur in conserved regions. Few rRNA modifying enzymes are known and their regulation is not well understood. At least one modifying enzyme, FtsJ, a specific methyltransferase which creates the 2'-O-methyluridine at U2552 in 23S RNA, has been identified as a heat shock protein.
The heat shock response is a mechanism which allows a cell to cope with a sudden increase in temperature or other environmental stress. The initiation of the heat shock response is transcriptionally regulated by an alternative sigma factor that directs RNA polymerase to different promoter sites which induces a rapid and transient expression of many genes that code for "heat shock proteins". Many of these heat shock proteins are chaperones and proteases, but many have unknown functions.
We are interested in using the sensitivity and specificity of new mass spectrometry-based methods to investigate the effect of the heat shock response on rRNA modification. In this study, Escherichia coli cells were cultured at 30 degrees Celsius to mid log phase and then the temperature was increased to a high but non-lethal temperature (42 degrees Celsius) to induce the heat shock response. Western blotting was used to measure the increase in DnaK, a major heat shock protein, thus verifying the induction of the heat shock response. Isolated rRNA from heat shocked and non-heat shocked cells is digested to nucleosides and the identities and relative amounts of modified nucleosides present in both samples are determined by HPLC and LC-MS approaches.
Keywords: RNA, Heat shock, LC-MS
Abstract:
Binding of guanosine substrate to the active site of group I intron has been investigated by kinetic and thermodynamic tools. Surprisingly, this intron binds guanosine very tightly, much tighter than the Anabaena and Tetrahymena group I introns. To explore why this intron might bind guanosine so tightly, we characterized the thermodynamics of G-binding by isothermal titration calorimetry. Guanosine binding by the Twort intron is enthalpically favored, contrasted by entropically favored guanosine binding with delta H =~0 in the other group I introns. To explain these differences, we hypothesize that the unbound conformation of the Twort ribozyme guanosine binding site more closely resembles bound one, which may be due at least in part to the presence of P7.1-7.2 subdomain.
We have also explored features of substrate recognition that are common to all group I introns. Recent crystallographic studies of the deoxyguanosine- and guanosine-bound group I intron ribozyme demonstrated that 2'-OH of guanosine substrate makes hydrogen bonds to 2'-OH of A119 (Golden et al., 2005; Adams et al., 2004). We hypothesize that this interaction helps to organize the structure of the active site. To test this, we generated a mutant ribozyme (Tw-dA119) having 2'-H substitution on A119, and determined the dissociation constant for guanosine of Tw-dA119. The Tw-dA119 bound at least 200-fold less tightly to guanosine, indicating that the active site of the ribozyme is not well organized without 2'-OH of A119. This is in agreement with crystal structures of group I introns which reveal the differences between guanosine-bound and deoxyguanosine-bound active site conformation and the inability of deoxyguanosine to serve as a substrate.
References:
Adams PL, Stahley MR, Kosek AB, Wang J, Strobel SA. 2004.Crystal structure of a self-splicing group I intron with both exons. Nature. 430:45-50.
Golden BL, Kim H, Chase E. 2005. Crystal structure of a phage Twort group I ribozyme-product complex. Nat Struct Mol Biol. 12:82-9.
Keywords: Group I intron, ribozymes, thermodynamics
Abstract:
The eight-nucleotide sequence of the 970 loop of helix 31 is conserved within the Bacteria, Archaea and Eukarya, but only A964, A969, and C970 are conserved among all three domains. The 970 loop also contains two of the eleven modified nucleosides in E. coli 16S rRNA, m2G966 and m5C967. Biochemical and structure studies have placed this loop near the P-site and have shown that it is involved in the decoding process and in binding the antibiotic tetracycline.
To identify functionally important nucleotides, sequence motifs and structural motifs in the 970 loop, all eight of the conserved loop nucleotides (964-971) were randomly mutated, selected, sequenced and assayed the viable mutants in our lab previously. Interestingly, single mutations at positions 966 and 967 produced hyperactive ribosomes. Biochemical and modeling studies suggest that initiation factor 3 (IF3) interacts with the stacked residues 966:967:968. Over-expression of IF3 specifically restores wild-type levels of protein synthesis to the 966 and 967 mutants. The 966 and 967 mutants, however, are not able to initiate translation from mRNAs containing CUG, AAG and ACG as the start codon. These data suggest that m2G966 and m5C967 are key components of IF3 binding to the 30 S subunit, but are not involved in discrimination of the initiator codon. To further determine the effect of those nucleotides in translational fidelity such as involvement in stop codons read-through, we tested all mutants of 966 and 967 with stop codons UAG and UGA in luciferase gene and it is shown that m5C967A and m5C967G showed the higher level of UAG stop codon read-through as compared to the wild type 16 S rRNA while as m2G966C showed higher level read-through in both UAG and UGA stop codons.
Keywords: Hyperactive mutants, Initiation factors, Translational fidelity
Abstract:
tRNA biogenesis has been thoroughly studied in bacteria andyeast, including transcription, maturation, and amino-acylation. Additionally important, tRNA genes themselves have been implicated in silencing of neighboring genes as well as being anchor points important in nuclear organization. In higher eukaryotes there is little known about how many tRNA genes are present, their expression, and what effect this has on local transcription and genomic organization. The primary focus of our research is the determination and verification of tRNA genes in the mouse genome.
We scanned the May 2004 Mus musculus genome with two tRNA detection programs, Aragorn (1) and tRNAscan-SE (2), that identify genomic sequences consistent with the conserved sequences and secondary structure of tRNAs. tRNAscan-SE searches for base pairing of clover leaf stems, poly-T RNA polymerase III (polIII) termination sequence, and A and B boxes which are polIII transcription factor recognition sites. Aragorn analyzes predicted secondary structure for tRNA homologies.
In order to verify the predictions, microarray chips were designed with multiple probes tiling each of the ~1,800 predicted tRNA genes. Fluorescently labeled RNA, from different mouse tissue and developmental stages, was used to probe the microarray. Over 500 of the predicted tRNAs were detected on the microarray. Confirmation of expression by Northern blot, comparative analysis of the different tRNA scanning programs, and analysis of the detected tRNAs will be presented.
References:
(1) Laslett, D. and B. Canback. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32: 11-16.
(2) Lowe, T.M. and S.R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955-964.
Keywords: tRNA, Mouse, bioinformatics
Abstract:
Formation of translation initiation complexes on leaderless mRNA occurs in the absence of signals upstream to the 5’-terminal start codon. Primer extension inhibition assays (toeprints) have shown that 70S ribosomes from E. coli bind leaderless mRNA more strongly than 30S subunits in vitro, suggesting a novel mechanism for the formation of translation initiation complexes on leaderless mRNAs. In the current study, leaderless mRNA containing the nucleotide derivative 4-thiouridine (4S-U) is used in site-specific crosslinking assays to identify leaderless mRNA/ribosome interactions that occur during translation initiation. Photoactivation with ultraviolet light stimulates 4S-U to form covalent crosslinks to nearby nucleotides and amino acids. Gel shift assays have shown that an oligonucleotide containing the first 20 nucleotides of the bacteriophage lambda leaderless cI coding sequence, with 4S-U at the +2 position (i.e., the U of the AUG start codon), binds 70S ribosomes and 30S subunits from E. coli in a tRNA-dependent manner. Photoactivation of 4S-U in complexes containing 70S ribosomes or 30S subunits produced crosslinks to rRNA and r-proteins exclusively within the small subunit. Crosslinks of the leaderless mRNA’s +2 position to 16S rRNA was localized to a single site at the 3’ end close to position 1530. Crosslinked r-proteins were affinity selected and identified by MALDI-TOF mass spectrometry. Of the r-proteins identified, S1, S3, S7 and S10/S18 displayed specificity for a 5’-AUG initiation codon. In accordance with the published crystal structure of the E. coli ribosome, it is likely that these r-proteins make contact with the 5’-AUG before the formation of the codon/anti-codon interaction between the 5’-AUG and initiator tRNA. This study will play a key role in understanding the mechanism by which these unique mRNAs are bound by ribosomes and translated into functional proteins.
Keywords: Translation Initiation, Ribosome, Leaderless mRNA
Abstract:
Bacterial mRNAs typically contain a 5’ untranslated region (5’ UTR) that includes a translation initiation region (TIR) which contains signals, such as the Shine-Dalgarno sequence and the spacer, that are important for 30S ribosomal subunit binding. Initiation factors (IFs) are thought to play a role in adjusting the initiation codon into the ribosomal P-site. “Leaderless” messages, in contrast, lack a 5’UTR and the traditional TIR. Current evidence suggests that translation initiation of leaderless mRNAs may follow a novel pathway involving 70S ribosomes. We are interested in the interactions that occur between leaderless mRNAs and the 30S subunit or 70S ribosome during translation initiation.
This study focuses on the role of IFs in ribosome binding of cI leaderless mRNA to 30S subunits or 70S ribosomes from Escherichia coli. Primer extension inhibition (toeprint) assays using 30S subunits suggest that IF2 increases stable binding while IF3 inhibits binding to cI mRNA. Alternatively, 70S ribosome binding is not altered by the addition of IFs. Filter binding allows for the measurement of less stable complexes; preliminary data suggests that IFs do not affect 30S or 70S association with cI mRNA.
To study interactions that occur between ribosomes and cI mRNA, a 20-nucleotide RNA oligomer that corresponds to the first 20-nucleotides of cI with a 4-thiouridine (4SU) at the +2 position of the start codon (cI-4SU), was used in crosslinking assays. UV-photoactivation of 4SU stimulates crosslinking to nearby nucleotides and amino acids. Crosslinks to 16S rRNA have been identified and localized close to 1530. Crosslinked ribosomal proteins have been identified by mass spectrometry and include S1, S3, S7, and S10/S18. These crosslinks were unaffected by the presence of IFs and/or initiator tRNA. Similar results were observed with 70S ribosomes. These mRNA/ribosome/IF interactions are important for understanding translation initiation of leaderless messages.
Keywords: translation, initiation factors
Abstract:
Streptomyces lividans ribosomes and subunits were isolated and washed with high-salt-buffer containing 1M ammonium chloride. These high-salt-washed 70S ribosomes (HS70S) and 30S subunits (HS30S) were characterized by toeprinting and filter-binding assays in the presence or absence of different combinations of purified Streptomyces initiation factors (IFs). Toeprint assays reveal that HS30S subunits exhibit tRNA-dependent binding to leaderless aph mRNA in the presence of IF2 alone or with IF1 and IF2 but not in the other possible combinations of IFs whereas HS70S ribosomes do not exhibit strong binding to leaderless aph in any possible combination of IFs. Additional analyses with filter binding assays revealed tRNA-dependent and -independent binding of HS70S ribosomes and HS30S subunits to leaderless aph mRNA with IF combinations that do not yield toeprint signals, suggesting that toeprint and filter binding assays differ with regards to the type, or strength, of complex detected. Additional characterization of the complexes is in progress, including competition assays to assess the importance of a 5’-terminal AUG in ribosome recognition of leaderless mRNA.
Keywords: Streptomyces, Translation initiation, Leaderless mRNA
Abstract:
Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of 5´ leader sequences of precursor transfer RNAs (pre-tRNAs). The enzyme is found in all phylogenetic domains; bacteria, archaea, and eucarya. The bacterial enzyme consists of a single RNA and one small protein. However, in the yeast Saccharomyces cerevisiae, nuclear RNase P is comprised of a single RNA and nine protein subunits. Each of the nine protein subunits, and the RNA subunit, are essential and it is not currently clear why the eukaryotic RNase P requires such a vast increase in its protein content. The proteins have potential functions in stabilizing RNA structure, substrate binding, substrate determination, roles in catalysis and localizing the holoenzyme.
In order to obtain information concerning the general structure of yeast RNase P we have initiated UV crosslinking studies with purified yeast nuclear RNase P holoenzyme and pre-tRNA substrate. To enable the detection of RNA-protein crosslinks and for purification purposes, nine yeast strains have been constructed where each of the individual proteins have been 6xHis tagged in parallel with the Rpr2 protein being TAP tagged. The results of this study will provide a framework for considering the overall architecture of the ribonucleoprotein and its interaction with pre-tRNA substrate.
Keywords: crosslinking, RNase P, tRNA
Abstract:
Fluorescence based techniques have proven very useful in studying the structure and function of proteins and nucleic acids in general. However, purifying fluorophore labeled proteins can be particularly challenging if the protein denatures easily and the labeling reaction is not 100% efficient. In order to overcome this challenge, we are developing an efficient method to separate fluorophore labeled proteins from unlabeled proteins using an RNA aptamer based affinity column specific to the fluorophore.
SRB 2 is a 54 nucleotide long RNA aptamer that has been previously evolved in vitro to bind sulforhodamine. [Wilson C., et al. (1998), Folding & Design 3:423] Previous work suggests that the aptamer selectively binds the fluorophore by recognizing both the planar aromatic ring system and the negatively charged sulfonate group.
Here, we show proof of concept for the use of SRB 2 in purifying tetramethylrhodamine (TAMRA) labeled proteins. Our fluorescence anisotropy measurements show that SRB 2 also binds TAMRA tightly and selectively (KD = 18 ± 8 nM) and this binding requires Mg2+ ions (K1/2 = 260 ± 100 nM). We have exploited this property to effectively release TAMRA from the aptamer by chelating Mg2+ ions in solution using only 5 mM EDTA. Finally, we have successfully labeled the 3’ end of SRB 2 aptamer with biotin and immobilized it on streptavidin-coated beads to purify rhodamine labeled proteins.
This method is of particular interest because it allows protein purification in very mild conditions. Furthermore, it can be easily expanded to purify proteins labeled with any fluorophore by simply changing the RNA aptamer.
Keywords: RNA Aptamer, Anisotropy, Tetramethylrhodamine
Abstract:
Protein synthesis by the ribosome is an essential process in all cells. The decoding region (helix 44) of 16S ribosomal RNA (rRNA) of the small subunit is a very important functional region; it is involved in binding to mRNA, tRNAs, and the large subunit rRNA. This decoding region is involved in translational accuracy, so that proteins are synthesized essentially without errors. This region is also responsible for translocation. In addition, some of the known antibiotics bind to the decoding region. There are three modified nucleosides in decoding site of E. coli 16S rRNA, namely 5-methylcytidine (m5C), 3-methyluridine (m3U), and N4, 2'-O-dimethylcytidine (m4Cm). The functional significance of those modified nucleosides is not completely understood. Out of the three modified nucleosides, m3U and m5C are commercially available. In contrast, m4Cm, one of the modified nucleosides in which both the base and sugar are methylated, must be prepared in laboratory. In order to carry out detailed studies on the roles of m4Cm and additional analogues, m4C and Cm, they were first chemically synthesized by a new method. Circular dichroism and 1D NOE NMR spectra for these nucleosides were compared with cytidine. Phosphoramidites of these nucleosides were prepared to construct a series of A-site/P-site (decoding region) RNA models. Biophysical studies were then carried out to determine the significance of the modified nucleosides within the context of the decoding region of 16S rRNA.
Keywords: modified nucleosides, decoding region, N4, 2-O-dimethylcytidine (m4Cm)
Abstract:
Splicing is an essential step in the maturation of eukaryotic pre-mRNA in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and a large number of proteins- that catalyzes the splicing process. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.
Here, we have used Fluorescence Resonance Energy Transfer and Single Molecule Spectroscopy to study the structural dynamics of a protein free U2-U6 complex from S. cerevisiae. Our FRET data clearly shows a Mg2+ induced large amplitude conformation change of the U2-U6 complex. In the absence of Mg2+ helix I and the U6-ISL are in close proximity, while in the presence of Mg2+ these two helices are far from each other. Our single molecule data shows that the conformational change consists of a two-step process, with an obligatory folding intermediate. We propose that the observed conformational change corresponds to the activation process expected to occur during spliceosomal assembly and catalysis. We are currently investigating this hypothesis by introducing point mutations in highly conserved regions of the U2-U6 complex.
Keywords: U2-U6 spliceosome RNA, single molecule, FRET
Abstract:
In the past, limited proteolysis has been employed to attempt to probe the structure of the ribosome with both 2D gel electrophoresis and nuclear magnetic resonance (NMR) as the detection step, with the purpose of determining information about structure of the ribosome in the absence of crystal structures. In this work, limited proteolysis has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allowing for simpler analysis with more reproducible results. In this study, time-dependent digestion was performed on Escherichia coli 30S ribosomal subunits; digestion of the ribosomal proteins allows for the determination of proteins that are resistant to proteolysis. The 30S ribosomal proteins were also analyzed in a variety of condition including their native form to account for the possible resistances to the experimental conditions. MALDI-MS provides accurate mass measurements of the digested proteins, and the resulting data allows for the characterization of surface-exposed regions of the RNP complex. This strategy reveals information about the surface regions more rapidly and from less sample as compared to the prior studies.
Keywords: Ribosome structure, MALDI-MS, Limited Proteolysis
Abstract:
The Polyadenylation of messenger RNA is essential for gene expression in eukaryotes. This process requires a complex of proteins, which have been characterized in mammals and yeast. The cleavage and polyadenylation specificity factor (CPSF) is indispensable for both cleavage and polyadenylation reactions. Orthologues of Cleavage and Polyadenylation Specificity Factor in Arabidopsis were identified and characterized by gene mutation, over- or under- expression. Unexpectedly, altered developmental and environmental responses were observed when normal expression levels of the genes were disturbed. These phenotypes include embryo lethal due to lack of female transmission of the AtCPSF73-II gene, male sterility due to delayed anther dehiscence by over-expression of the AtCPSF73-I gene. The gene encoding the 30 Kda subunit of CPSF is non-essential in Arabidopsis, mutant of which increases oxidative stress tolerance seemingly through altered responses of the protein to calmodulin. These results suggest the regulatory roles of mRNA polyadenylation that are beyond normal function in house-keeping.
Keywords: CPSF
Abstract:
The 3’-end cleavage and polyadenylation of eukaryotic pre-mRNA involves several protein complexes containing more than a dozen of subunits. Yeast (S. cerevisiae) Clp1p is one of these subunits and is essential for appropriate 3’-end processing. yClp1p interacts with Rna14p, Rna15p and Pcf11p subunits, forming the CFIA complex of polyadenylation machinery. The biochemical and biological function of yClp1p is thought to be largely conserved between yeast and mammals. However, little is known about plant counterpart of yClp1p. We have identified a small gene family of Arabidopsis homologous of yClp1p and other components of CFIA. To elucidate the significance of these Arabidopsis proteins in polyadenylation and transcription termination, we have tested their interaction with other polyadenylation factors using yeast two-hybrid and in vitro pull-down assays. Analysis of T-DNA knockouts of Arabidopsis genes encoding homologous of yClp1p revealed interesting phenotypes, in which the development of the embryos are impacted. The significant of the finding will be discussed.
Keywords: Clp
Abstract:
Pseudouridine is the most abundant and common posttranscriptional modification found in RNA. Pseudouridines are found at specific locations in the rRNAs and tRNAs of Archaea, Bacteria and Eukarya. However the function of pseudouridines on a molecular level remains less understood. The localization and identification of pseudouridines in RNA is required in order to decipher their functional role. Appropriate experimental techniques have been developed for the identification of pseudouridines but these are often labor-intensive and time and sample-consuming.
In this work, we describe a mass spectrometry-based approach for the detection of pseudouridines in RNA. In our approach, the RNA is first digested into fragments by the endonucleases, RNase A or T1. Mass spectral data of the digest fragments are obtained and serve as the control. The digest products are then subsequently derivatized with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC). Pseudouridines are identified by a direct comparison of the control (non-derivatized digest fragments) to their CMC-derivatized counterparts. After CMC derivatization, pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry.
Here, we demonstrate the application of this approach to pseudouridine identification in a mixture of Escherichia coli tRNAs. Current efforts are focused towards the detection of pseudouridines in larger RNAs, such as the 23S rRNA of E. coli. We find that this approach enables the rapid screening for pseudouridines in both small and large RNA systems.
Keywords: pseudouridine, mass spectrometry, endonuclease
Abstract:
The ribosome is a known target for antibiotics, which bind to a limited number of sites, in both the large and the small ribosomal subunits.(1) One important target is the A site of the 16 S rRNA of the 30 S subunit. The universality of the decoding phenomenom renders the A site an interesting target, with reduced possibilities for mutations. In our research, diverse modes of selection were used to identify peptides that bind to the 27-nucleotide A-site RNA model,(2) including phage display and chemical split-pool peptide library synthesis. Biophysical characterization of the selected peptide sequences was done using several methods, such as fluorescence spectroscopy, electrospray-ionization mass spectrometry, and enzymatic footprinting. These studies give some insights about the affinity and specificity of the peptides with their target RNA. Understanding these intreactions will lead us to modify the peptides to enhance their affinity and specificity, therefore their activity.
References:
1. Steitz. T. A., On the structural basis of peptide-bond formation and antibiotic resistance from atomic structures of the large ribosomal subunit. FEBS Letters 2005, 579, 955-958.
2. Fourmy D., Blanchard S. C., Puglisi J. D., Science 274, 1367 (22 november, 1996).
Keywords: rRNA, peptides
Abstract:
Ribonuclease P holoenzyme (RNase P) catalyzes the 5' maturation of precursor tRNAs (pre-tRNAs). The B. subtilis RNase P holoenzyme consists of an RNA component and a small protein component. The RNA component of RNase P alone is catalytically active in vitro; however the protein cofactor is essential for activity under physiological conditions. The protein component directly interacts with the 5' leader of the pre-tRNA substrate and enhances metal affinity. While metal ions capable of inner-sphere coordination are not essential for formation of the RNase P holoenzyme•pre-tRNA complex, inner-sphere coordination of metal ions is essential for catalytic activity. Defining the roles of metal ions in catalysis by RNase P holoenzyme is essential to understand its mechanism.
We are using fluorescence titration and transient kinetics to probe the thermodynamics and kinetics of binding Ca(II) to the RNase P holoenzyme•pre-tRNA (ES) complex, and time-resolved FRET to characterize structural alterations in a Ca(II)-induced conformational change in this complex. In 2 mM Co(NH3)63, 189 mM KCl, and 50 mM Tris/MES, pH 6.0, addition of 100 μM CaCl2 enhances the binding affinity of pre-tRNAAsp for B. subtilis RNase P holoenzyme by 3-4 fold. Fluorescence stopped-flow and trFRET data suggest that Ca(II) binding induces a conformational change in the ES complex. Addition of similar concentrations of Mg(II) also induces this conformational change, but does not significantly enhance cleavage activity. These data suggest that formation of the RNase P holoenzyme•pre-tRNA complex creates a high affinity, inner sphere binding site for Ca(II) or Mg(II). Furthermore, metal binding is coupled to a conformational change that enhances substrate binding affinity and organizes the active site in the ES complex. However, one or more additional Mg(II) ions are required to activate catalytic activity.
Keywords: RNase P, conformational change, metals
Abstract:
RNase P is an enzyme that catalyzes the 5' maturation of precursor tRNA, generating a mature tRNA and a short 5' leader RNA. Specifically, RNase P derived from Bactillus subtilis contains P RNA which is the catalytically active RNA component and a small protein cofactor known as P protein. Our lab has been researching the kinetic mechanism for this enzyme by fluorescence FRET stop flow techniques. The product release is rate limiting in steady-state conditions for the cleavage reaction catalyzed by both PRNA and the RNase P holoenzyme. In PRNA catalyzed reaction, the leader is released before the mature tRNA. However, in the holoenzyme-substrate complex, the leader may interact with the P protein and the mature tRNA with the P RNA which may result in a different product release sequence.
To study this question, we have directly monitored the dissociation rate constants for the two products from the RNase P holoenzyme: 5' leader and the mature tRNA during single turnover reaction to see which end disassociates faster to differentiate between at least two different possible pathways for product dissociation. We have prepared pre-tRNA substrates with fluorescence labeling at either 5' leader or the 3' tRNA moiety and measured the dissociation rate constants of both products by fluorescence stop flow techniques. Side-by-side comparison between the product release rates for the 5' leader and the mature tRNA for the RNase P holoenzyme will be studied also as a function of the leader length to understand the effects of protein-leader interaction on product dissociation kinetics.
Keywords: RNase P, Transient kinetics, Fluorescence
Abstract:
The coupling of high performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) has proven to be a formidable technique for the separation and analysis of oligonucleotide mixtures. The use of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) to analyze and quantify isotopically labeled ribonucleic acids (RNAs) has provided the ability to analyze defined and known mixtures; however, the analysis and quantification of complex mixtures is still limited by this technique. Capillary LC coupled to ESI-MS is currently under development in order to separate and quantify complex mixtures of RNA. For method development, incorporation of the isotope is performed by digestion of a known RNA in the presence of H216O or H218O. Labeled oligonucleotide fragments are then separated with ion pairing chromatography using triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol as ion pairing agents. Mass spectrometric analysis reveals the relative abundance of 16O and 18O labeled oligonucleotides. Although current method development has been performed with Escherichia coli (MRE 600), this method will be applicable to biological systems wherein RNA abundances vary due to growth or other environmental conditions.
Keywords: Mass Spectrometry, High Performance Liquid Chromatography
Abstract:
Exosome complexes are 3’ to 5’ exoribonucleases comprised of subunits that are essential for viability, conserved in all eukaryotes and archaea, and critical for numerous distinct RNA metabolic pathways. Despite much progress in defining the RNA substrates of the exosome, the exact mechanisms underlying how the exosome either processes or degrades these substrates remains unclear. Genetic and biochemical studies in yeast suggest that exosome subunits assemble into and function in multiple distinct exosome sub-complexes. Our recent cell biological studies show that exosome subunits differentially distribute in Drosophila melanogaster tissue culture cells. We are thus testing the following hypothesis: differential exosome subunit subcellular localization reflects distinct exosome subunit subcomplexes with correspondingly specialized functions and RNA substrates.
Keywords: exosome, RNAi, RNA turnover
Abstract:
Bcl-2-like transcription factor (Btf) is a novel nuclear speckle protein identified in a proteomic analysis of purified nuclear speckles. Btf exhibits a subnuclear localization that is unique compared to that of other nuclear speckle proteins. Immunofluorescence localization of Btf shows that it is not completely coincident with other nuclear speckle proteins, and it is not largely enriched in the “core” of the speckles. Rather, Btf is enriched in regions near the periphery of nuclear speckles. While nuclear speckles are sites of enrichment for pre-mRNA processing factors, most transcription and pre-mRNA processing occurs outside of the speckles. In addition to the peripheral nuclear speckle localization, Btf is also found in a punctate nucleoplasmic distribution that is similar to the foci seen when transcription sites are labeled by bromo-UTP incorporation. Btf is not yet characterized with regard to the function of this novel localization, but it is important to determine if the localization corresponds with transcription sites. Our data shows that Btf can accumulate at an activated reporter gene locus. We hypothesize that Btf plays a role in transcription and/or pre-mRNA splicing, and that the novel Btf localization reflects this function.
Keywords: transcription, splicing, nuclear speckles
Abstract:
Proximal Spinal Muscular Atrophy (SMA), the leading genetic cause of infant mortality in humans, is caused by deletion or mutation of the survival of motor neuron gene-1 (SMN1) (Lefebvre et al. 1995; Jablonka et al. 2000). Homozygous deletion of the homologous gene (SMN) in mouse models leads to early embryonic lethality and thus fails to adequately model the human disease. It has been shown that human SMN2, a nearly identical gene, rescues the mouse knock-out phenotype in a dose dependent manner, where intermediate doses of SMN2 lead to a muscular atrophic phenotype (Hsieh-Li et al. 2000; Monani et al. 2000). Because the human SMN2 gene contains a point mutation that leads to inefficient splicing of the gene, it is thought that the SMN2 gene acts like a hypomorph and produces only enough levels of protein to support embryogenesis, but not the development and maintenance of healthy nerves after birth. Correction of SMN2 splicing is therefore a therapeutic option to reinstate SMN activity in SMA patients. However, the timing of therapeutic intervention will be important. Studies in zebrafish predict that restoration of SMN function during embryogenesis may be important for axonal pathfinding, while the mouse models and normal human disease progression suggest that post-natal treatment may be sufficient for amelioration of the disease phenotype. We have designed a temporally inducible-SMN transgenic mouse model that can be used in the SMA disease model background to determine the timing of potential SMN replacement therapies. In our inducible Smn mouse, wild-type SMN expression is Cre-inducible and will be controlled by adding a drug, tamoxifen. It is our immediate future plan to determine the therapeutic window for SMN replacement. To do this, we will cross currently available SMA mouse models the inducible Smn mouse model.
Keywords: Spinal Muscular Atrophy, Alternative Splicing, Mouse Model
Abstract:
Of the nearly 100 known post-translational modifications of RNA, the most common is the conversion of uridine (U) to pseudouridine (Ψ). The enzymes responsible for this conversion, the Ψ synthases, are a superfamily of enzymes that share no global sequence similarity yet share the same core β-sheet fold. Crystal structures of representative members of each family have been solved, most recently RluA in complex with a stem-loop RNA (Hoang, C., et al., and Ferré-D'Amaré, Mol Cell, in press). Biochemical studies have shown that RluA is potently inhibited by RNA containing 5-fluorouridine, and RluA and the inhibitory RNA co-migrate on both SDS-PAGE and urea-PAGE gels, suggesting a covalent protein-RNA adduct. However, no such covalent bond is observed in the cocrystal structure. A variety of spectroscopic and biochemical techniques have been employed to probe for a covalent linkage between RluA and 5-fluorouridine in RNA, and the results are presented here.
Keywords: pseudouridine synthase, RluA
Abstract:
Ribonuclease P (RNase P) is a ubiquitous and essential ribozyme that cleaves the 5' leader sequence of precursor-tRNA to yield mature tRNA. RNase P is also a ribonucleoprotein complex, consisting of a large RNA subunit and a number of associated proteins that are necessary for activity in vivo. Although the RNA portion of the enzyme is conserved between the domains, the protein content varies greatly: one protein in bacteria; 4 or 5 proteins in archaea; and 10 proteins in eukarya. Interestingly, the archaeal proteins are homologous to a subset of the eukaryal proteins, yet neither domain contains a protein with sequence similarity to the single bacterial RNase P protein. An inverse correlation between RNA proficiency and associated protein content can be observed when comparing the domains of life: the RNA is active alone in vitro in the protein-sparse bacteria; active alone in only some archaea; and has not been shown to be active without the proteins in the protein-heavy eukaryal RNase P.
Recent efforts in our lab and others' have elucidated the three-dimensional structures of the individual protein subunits associated with archaeal RNase P, but the enticing question remains: how do these proteins confer an activity boost to the RNA? In bacteria, the single protein's role is relatively well-understood, but in archaea and eukarya the function of the multiple proteins is still unknown. It has been hypothesized that the proteins may aid substrate binding and specificity, or alter the structure of the RNA to stabilize an active conformation.
To address this question, we will investigate the structure of RNase P from Pyrococcus furiosus (Pfu). Instead of tackling the entire holoenzyme, we have begun working with a ribonucleoprotein complex comprising a truncated mutant of the RNA subunit (containing the catalytic portion of the molecule) and the proteins Pop5 and Rpp30. A new protocol has been developed to prepare large-scale amounts of this complex, and it has been confirmed to be catalytically active. Mass spectrometry will be employed to confirm the stoichiometry of the subunits within the complex, and crystallographic screens are underway.
Keywords: RNase P, ribonucleoprotein, structural biology
Abstract:
Glutamyl-prolyl tRNA synthetase (GluProRS), the bifunctional aminoacyl tRNA synthetase, is an integral component of the tRNA multisynthetase complex (MSC) containing 7 other tRNA synthetases and 3 non-synthetase proteins. In addition to aminoacylation of glutamic acid and proline to cognate tRNAs during protein synthesis, GluProRS also has a regulated, noncanonical activity in translational control. In human monocytic U937 cells, upon activation with gamma-interferon (IFN), GluProRS is phosphorylated and interacts with 3 other proteins to form the Gamma-interferon Activated Inhibitor of Translation (GAIT) complex. The GAIT complex binds to the 29-nucleotide GAIT element in ceruloplasmin (Cp) mRNA and blocks its translation. Here we investigate the mechanism of activation of GluProRS by phosphorylation, and the role of GluProRS phosphorylation in the formation of the GAIT complex. IFN induces rapid phosphorylation of GluProRS at serine residues in the linker domain that joins the two catalytic domains of GluProRS. Immunoaffinity purification, coupled with mass spectrometry, and site-directed mutagenesis has revealed Ser886 and Ser999 as the stimulus-dependent phosphorylation sites. In vitro studies have shown that phosphorylation of the two sites is independent of each other and induced by kinases belonging to two distinct groups. Time course studies suggest that Ser886 phosphorylation precedes phosphorylation of Ser999. Bioinformatic analysis, together with mutation and kinase inhibitor studies has revealed SPXR, a target of proline-dependent Ser/Thr kinases, as the critical kinase recognition motif required for phosphorylation of Ser886. Protein kinase C is a candidate for phosphorylation of Ser999. Phosphorylation induces the release of GluProRS from its usual residence in the MSC and is required for interaction with other protein components to form the active GAIT complex. Thus, two-site serine phosphorylation of GluProRS is required for its translocation from the MSC to the GAIT complex, thereby regulating transcript-selective translational silencing in inflammatory macrophages.
Keywords: GAIT, Phosphorylation, Glutamyl-proly tRNA synthetase
