RRM
2008 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract not available online - please check the printed booklet.
Abstract:
The ribosomal protein L13a inhibits the translation of IFNã-induced ceruloplasmin (CP). Upon release from the 60S ribosome, L13a forms a GAIT (IFN-gamma activated inhibitor of translation) complex, which binds to the 29 nucleotide sequence, refered to as GAIT element, on the 3’UTR of CP mRNA for silencing. Thus following initial upregulation of CP there is a shut-off of CP after 16 hrs. We hypothesize that the translational silencing mechanism is not exclusive for CP but applies to multitude of proteins. Therefore, we conducted a genome-wide analysis using Affymetrix Genechip and Inflammation Responsive Genearray to identify other proteins. Following 0, 4, and 18 hr. IFNã treatment, polysome bound mRNA (translation active pool) and unbound mRNA (inactive pool) were isolated using sucrose gradient and processed. We focused on mRNAs that had a similar polysomal profile as CP, i.e. translationally active at 4 hr., while inactive at 18 hr. A cluster of mRNAs encoding different chemokines and chemokine receptors and molecules important in cytokine signaling was identified as common hits in the two independent approaches. The selected mRNA targets were validated using real-time PCR. Further, in silico-determined GAIT element in the 3’UTR of the selected mRNAs were confirmed as functional cis-acting elements for translational silencing by luciferase reporter assay. The newly identified target mRNAs also required L13a for silencing. Thus this cohort of mRNAs may represent a new inflammation responsive post-transcriptional operon regulated directly at the level of translation in order to control inflammation.
Keywords: translation control, microarray, IFN gamma
Abstract:
The PMR1 mRNA endonuclease catalyzes the selective decay of a limited number of mRNAs. It participates in multiple complexes, including one containing c-Src, its activating kinase, and one containing its substrate mRNA. We used tandem affinity (TAP) chromatography to identify proteins in HeLa cell S100 associated with the mature 60 kDa form of Xenopus PMR1 (xPMR60). Unexpectedly this identified a number of cytoskeleton-associated proteins, most notably the Ena family proteins mammalian Enabled (Mena) and vasodilator stimulated phosphoprotein (VASP). These are regulators of actin dynamics that are concentrated at the leading edge in lamellepodia. We show that only a portion of xPMR60 interacts with Mena and VASP in vivo, and that this interaction is independent of the cytoskeleton and RNA. Mena interacts with both c-Src and xPMR60 and sediments with xPMR60 on glycerol gradients in the functional complex containing xPMR60 and its substrate mRNA. To address the function of the xPMR60-Mena interaction, wound healing experiments were performed and showed that cells expressing xPM60R have a more invasive phenotype than those not expressing xPMR60. Confocal microscopy was used to look at the interaction of PMR, actin and VASP in cells. Our results point to an interaction of xPMR60 with the Ena family proteins that localizes the decay of specific mRNAs at the leading edge of the cell.
Keywords: PMR, cytoskeleton-associated proteins
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Recent studies have indicated that while 93% of the human genome is transcribed into RNA, only 2% of it codes for proteins. It is estimated that human genome contains 70,000 large non-protein coding transcripts; however, the functional mechanism of this novel class of cellular regulators is almost completely unknown. We have analyzed the cellular function of one such RNA, the BORG RNA, which was originally described as a BMP2-responsive gene (Takeda et al., 1998). BORG is a 2766 nucleotide long transcript and is both spliced and polyadenylated. Phylogenetic analyses proved that the RNA is found in several mammalian genomes including human, chimpanzee, macaque, cow, pig, dog, and rat in addition to mouse, with ~ 65% conservation between human and mouse in a conserved region in its third exon.
Analysis of BORG expression in mouse tissues indicated that it is highly expressed in neural tissues. To determine the role of BORG in cellular differentiation, we took advantage of mouse C2C12 pre-myocytic cell line. BORG is expressed in C2C12 cells at a low level. We obtained stable cell lines that overexpressed full-length BORG and induced muscle differentiation to determine if BORG overexpression affects their ability to differentiate. Surprisingly, rather than differentiating into muscles, the cells developed round cell bodies and long branched processes that interconnected to form a network, features characteristic of neuronal progenitor cells. Wild type cells, those transfected with empty vectors and BORG shRNA knockdown cells did not display this phenotype when received differentiation media, rather, they showed phenotypic changes characteristic of muscle differentiation. Immunostaining and RT-PCR assays on differentiated BORG overexpression cells indicated that they express neuronal markers TUJ1, while neither the precursor cells nor BORG shRNA knock down cells express these markers. The differentiated cells had lost the ability to divide, consistent with neuronal differentiation. The ability of a large non-coding RNA to reprogram muscle precursor cells, which are of mesodermal origin, to neuronal cells, which are of ectodermal origin, provides an example of the crucial, and hitherto unknown, roles played by non-coding RNAs in regulation of cellular function and development.
Keywords: non-coding RNA
Abstract:
The TRAMP complex is critical for quality control and 3’ end processing of nuclear RNA in S. cerevisiae. The TRAMP complex, which consists of the poly(A) polymerase Trf4p, the RNA binding protein Air2p, and the DExH RNA helicase Mtr4p, appends short poly(A) tails to RNAs designated for degradation by the nuclear exosome. Both the 5’ to 3’ polyadenylation activity of Trf4p, and the 3’ to 5’ helicase activity of Mtr4p are required for TRAMP to stimulate RNA degradation by the exosome. However, it is unclear how these two activities, given their opposing directionality, function together in one complex. To illuminate this question we have quantitatively analyzed polyadenylation and RNA unwinding activities for a reconstituted, purified TRAMP complex. Using a newly developed method that allows us to measure rate constants for addition of individual adenylate residues to the RNA, we show that poly(A) addition does not occur in equal steps but in a characteristic pattern where RNA species with 4-10 A residues accumulate. Functional Mtr4p is required for this characteristic pattern; it is not observed for TRAMP with non-functional Mtr4p, or in the absence of Mtr4p. Interestingly, we find that TRAMP requires a minimal number of 4 unpaired nucleotides to unwind RNA duplex substrates, suggesting that the polyadenylation by Trf4p serves to provide a landing site for Mtr4p, which then unwinds RNA secondary structure. We further show dramatic stimulation of the helicase activity of Mtr4p by Trf4p/Air2p, with a preference for duplexes with short overhangs. Collectively, our data indicate strong functional interdependence of Trf4p and Mtr4p during polyadenylation and duplex unwinding by TRAMP. The opposing polarities of polyadenylation and unwinding activities are critical for RNA recognition and ensure that only a minimal number of adenylates are added to RNAs prior to further processing.
Keywords: poly(A) polymerase, helicase
Abstract not available online - please check the printed booklet.
Abstract:
In the maturation process of a eukaryotic messenger RNA (mRNA) several steps need to be completed if proper gene expression and fidelity are desired. A particularly important step in this pathway is splicing, which involves the removal of intervening sequences (introns) from the precursor messenger RNA (pre-mRNA). This process involves two transesterification reactions catalyzed by a multimegadalton RNA-protein complex termed the spliceosome1. The spliceosome is composed of 5 ribonucleoproteins (RNPs) which contain a total of over 100 different proteins, and 5 small nuclear RNAs (snRNAs)2. The architectural complexity of the spliceosome is only overshadowed by the network of protein-protein, protein-RNA, and RNA-RNA interactions which the spliceosome utilizes to catalyze the splicing reaction. To fully understand the complexity of the interactions between spliceosomal components and pre-messenger RNAs, it is necessary to observe the process in a manner that preserves dynamics without disrupting the transient interactions that are crucial to splicing. Working in collaboration with John Abelson (UCSF) we have developed a single molecule FRET (sm-FRET) splicing assay. By using pre-mRNAs fluorescently labeled with a FRET pair, and Total Internal Reflection Fluorescence (TIRF) microscopy we have tracked the conformational states through which the spliceosome takes a single pre-mRNA. With the ability to track all the steps in splicing, from recognition to catalysis, we can start asking questions that have been difficult to answer with traditional biochemical techniques. Preliminary FRET data has shown us that at every step of the reaction the pre-mRNA substrate is taken through a variety of reversible conformational states. The use of temperature sensitive spliceosomal components and pre-mRNAs with mutations at conserved sites will allow us to assign particular conformational states to the different steps in splicing and ultimately determine the kinetics of spliceosome assembly and catalysis.
References:
(1) E. Brody, J. Abelson. Science. 1985 May 24;228(4702):963-7
(2) JP. Staley, C. Guthrie. Cell. 1998 Feb 6;92(3):315-26.
Keywords: single molecule, splicing, FRET
Abstract:
We previously showed that protein-free fragments of human U6 and U2 snRNAs performed a two-step splicing reaction on short RNA substrates. To determine if the ability to catalyze splicing without the help of proteins is a phylogenetically conserved feature of the spliceosomal snRNAs, we performed a detailed analysis of the structural and catalytic features of U6 and U2 snRNAs derived from human and yeast. The genetically deduced secondary structure of the yeast U6/U2 complex is different from that in human. Also, yeast U6 snRNA shows divergence from the consensus sequence in a number of residues of the catalytically critical central domain. While the RNAstructure algorithm predicts a similar global base-paired structure for human and yeast U6/U2 complexes, a number of structural elements, such as U6/U2 helices I and III, and the U2 stem I are predicted to fold differently.
In order to study the catalytic properties of the yeast snRNAs, we first determined if yeast U6 and U2 can perform protein-free splicing. Although the snRNAs could bind the splicing substrates, they did not show catalytic activity under any of the conditions tested, while human U6/U2 was active. To determine whether this resulted from the differences in sequence or secondary structure of the yeast and human snRNAs, we made small, directed mutations in yeast U6 and U2. These mutations induced individual domains of the yeast U6/U2 complex to form basepairing interactions similar to those predicted to form in human by RNAstructure algorithm and genetic analyses. A triple point mutation in the helix II region was introduced to stabilize this helix. Two point mutations in helices I and III induced these two helices to form a structure similar to that predicted to form in human. Interestingly, while introduction of the helix II or helix III mutations did not result in catalytic activity, modification of helix I led to a weak activity in protein-free splicing assays. A triple mutant that contained the helix II and III mutations in addition to the helix I point mutation showed enhanced activity. These results underscore the crucial contribution of the secondary structure of the U6/U2 complex to catalytic function and further indicate that the ability to perform protein-free splicing is phylogenetically conserved from yeast to human.
Keywords: U6U2, Splicing, Catalysis
Abstract not available online - please check the printed booklet.
Abstract:
The increasing number of resistant bacteria to the existing antibiotic panoply is of great concern. Among these antibiotics, almost half interfere with the protein synthesis machinery, whose core is the ribosome. Within its two subunits, the ribosome harbors several sites targeted by antibiotics (1), one of them being the A site. The A site, located on helix 44 of the small subunit 16S rRNA, is universally responsible for decoding mRNA into protein. In our research, a peptide binding to a 27-nucleotide A-site rRNA model (2) was identified by phage display. The role played by each amino acid in the affinity and specificity of the binding to the A-site rRNA target was investigated by performing alanine screening, in which the variant peptides were studied using biophysical methods such as electrospray-ionization mass spectrometry, enzymatic footprinting, and circular dichroism. The insights gained regarding interactions of the variant peptides with the E. coli A-site rRNA will lead us to modify the peptide to enhance its affinity, specificity, and activity as a potential antibiotic.
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, (4), 955-958.
(2) Fourmy D., R. M. I., Blanchard S. C., Puglisi J. D., Structure of the A Site of Escherichia coli 16 S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic. Science 1996, 274, (5291), 1367-1371.
Keywords: Ribosome, Antibiotic, Peptide
Abstract:
Reverse transcription of the human immunodeficiency virus type-1 (HIV-1) single-stranded RNA genome into double-stranded cDNA requires a complex series of extensions, jumps, and template switches. Reverse transcriptase uses a host cell tRNALys,3 primer, which must be unwound and annealed onto the viral RNA at a complementary primer binding site. In vitro, the restructuring of these RNAs is greatly enhanced by the presence of the potent nucleic acid chaperone, HIV-1 nucleocapsid protein (NC). However, in vivo evidence suggests that the Gag poly-protein precursor is responsible for facilitating tRNA annealing. Before proteolysis, Gag consists of matrix (MA), capsid (CA), spacer 1 (p2), NC, spacer 2 (p1), and p6 domains. Previous studies demonstrated that Gag can facilitate tRNA annealing in vitro; however, the contribution of each domain to its nucleic acid chaperone activity is unknown. In this work, we show that Gag mediates tRNA annealing at a reduced rate relative to HIV-1 NC. The NC domain is essential for Gag-mediated annealing, while the MA domain appears to inhibit Gag’s chaperone activity. Whereas WM-Gag, a monomeric variant harboring mutations at the dimerization interface within CA, displays similar annealing rates as wild-type Gag, Gag variants containing NC but lacking MA (i.e., CANC) show elevated rates of tRNA annealing. Interestingly, inositol phosphates (IPs), which are known to bind to basic residues K30 and K32 within MA and modulate Gag particle assembly in vitro, stimulate the chaperone activity of Gag. Stimulation by IPs was shown to depend on the presence of MA residues K30 and K32, and the maximum effect was achieved at a 1:1 Gag:IP ratio. Taken together with previous data, these results suggest that IP or membrane binding by the MA domain results in a conformational switch that stimulates Gag’s ability to facilitate annealing of the tRNA primer via the NC domain. This work was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Keywords: HIV, reverse transcription, RNA chaperone
Abstract:
MicroRNAs (miRNA) are short 20-25 nucleotide RNA molecules that negatively regulate gene expression in animals and plants. We have developed a vector-based genetic library including minigenes for most known human miRNAs. This library provides a widely applicable resource for gain-of-function studies for miRNA and will significantly facilitate our studies to identify novel miRNA function. In this work, we have designed a dual-reporter method to investigate miRNA functions in the NF-ƒÛB signaling pathway in conjunction with our genetic library. A firefly luciferase gene (luc) under the control of a minimal CMV (mCMV) promoter, which is downstream of transcription response elements (TRE), is integrated into the chromosomes of a host cell. The mCMV promoter is only activated when a specific transcription factor (TF) (for example NF-kappaB) binds to the TRE. We use lentiviral-based TF vector to integrate the first reporter into the host chromosomes. The second reporter Renilla luciferase (Rluc) upstream of the 3'-UTR sequence from the TF gene is constitutively expressed. With miRNAs directly targeting the TF gene, both reporters will be down-regulated. However, if miRNA indirectly down-regulates the TF, only the first reporter expression will be affected. Similarly, miRNAs targeting any gene that modulates the TF function can be screened with its 3'-UTR downstream of the second reporter. Coupled with our vector-based lentiviral miRNA genetic library, we are able to investigate whether any miRNA is relevant to a signaling pathway rather than whether one miRNA down-regulates one particular gene. We have identified one miRNA from the above assay that upregulated NF-kappaB-dependent reporter expression the most. We then studied the target of this miRNA, which inhibits NF-kappaB activation. Moreover, the transcription of this miRNA is controlled by NF-kappaB. Disclosing this miRNA in the NF-kappaB pathway would present a gene regulatory mechanism for chronic or persistent NF-kappaB activation by TNF-alpha, in which the miRNA mediates cellular NF-kappaB signaling through a positive feedback loop. These results may also offer clues to NF-kappaB activation in some tumors and provide novel biomarkers for cancer diagnosis.
Keywords: microRNA, NF-kappaB, signal pathway
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:
The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA motif found on the genomic and antigenomic strands of the HDV RNA and within the mammalian transcriptosome. The cleavage reaction is believed to proceed through a general acid/general base mechanism in which C75 serves as a general acid and a magnesium-bound water serves as the general base. Naturally occurring variants of the HDV ribozyme contain a semi-conserved G•U wobble base pair at the cleavage site. We propose that the N7 of cleavage site guanosine (G1) provides a ligand to the catalytic metal ion. The evidence for this includes: 1) the deleterious effect of a 7-deaza modification at G1 on catalysis, 2) apparent reduction in magnesium affinity resulting from a 7-deaza modification at G1, 3) observation of magnesium binding and guanine N7 coordination upon C75 deprotonation in Raman spectra, and 4) failure to observe the Raman features corresponding to magnesium binding and guanine N7 coordination when a 7-deaza modification is introduced at G1. Together with previous biochemical studies, a model of the catalytic metal ion bound within the HDV active site is proposed. This model explains the solution kinetic and Raman spectroscopy results presented here and is in agreement with published biochemical data on the wild-type HDV ribozyme.
Keywords: Hepatitis delta virus (HDV) ribozyme, Raman spectroscopy, 7-deazaguanosine
Abstract:
Assembly and processing of the small ribosomal subunit (40S) in yeast requires multiple steps and many associated factors, as the construction of this essential piece of metabolic machinery is closely regulated. Cleavage steps of the precursor ribosomal RNA (rRNA) are strictly ordered, though the regulation of this ordering remains only partially elucidated. Nob1 is an essential, conserved protein involved in ribosome biogenesis in Saccharomyces cerevisiae. Specifically, cells depleted in Nob1 show a defect in the final step of processing of the 40S small ribosomal subunit rRNA: Cleavage of 20S pre-rRNA to form the mature 3’ end of 18S rRNA does not occur (1). Because Nob1 contains a predicted PIN domain that is required for this cleavage (2), it has been proposed that Nob1 is the endonuclease responsible for maturation of the 20S rRNA to 18S rRNA. However, Nob1 binds the rRNA precursor at a very early stage, and the mechanism by which the endonuclease activity is regulated to only cleave at the final step remains unknown.
Here, we show that recombinant Nob1 binds to rRNA fragments from the 3’ end of the 20S rRNA. Nob1 binds ten-fold more tightly to a substrate (20S)-mimic than product (18S)-mimic. Analysis of Nob1 affinities for a collection of rRNA fragments indicates that sequences upstream and downstream of this region cooperate in Nob1 binding. Structure probing with DMS indicates that these RNAs fold into different conformations in the absence of Nob1. Additionally, Nob1 protects a different subset of residues in these RNAs. These results suggest that conformational changes in the RNA regulate Nob1 binding and activity in generating the mature 3’ end of 18S rRNA.
References:
(1) Fatica, A., Oeffinger, M., Dlakic, M., and Tollervey, D. (2003) Mol. Cell. Biol. 23, 1798-1807.
(2) Fatica, A., Tollervey, D., and Dlakic, M. (2004) RNA 10, 1698-1701.
Keywords: ribosome assembly, conformational change, endonuclease regulation
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. Our LASR experiments indicate that RNA kissing complex is a stable intermediate state that facilitates the dimerization reaction.
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, refolding, RNA
Abstract:
Linear dsDNA viruses, including bacteriophage phi29, translocate their genome into a pre-assembled procapsid to near-crystalline density. DNA packaging motor of phi29 uses dodecameric connector as a DNA entry channel, hexameric packaging RNA (pRNA) and packaging enzyme gp16 to drive force to overcome the unfavorable packaging reaction.
Biochemical studies showed that 5’/3’ paired helical region of pRNA in the pRNA/procapsid complex is for gp16 binding, while its central domain binds to the procapsid. pRNA bulge C18C19A20 that is essential for DNA packaging was dispensable for gp16 binding. The interaction of gp16 with nucleic acids was also investigated using recently developed single molecule TIRF system. The study showed that gp16 binds nucleic acids (RNA or DNA) depending on their structures (single-stranded or double-stranded) and chemistry (purine or pyrimidine). Gp16 contains a conserved nucleotide triphosphate binding motif. The steady-state analysis showed that pRNA or DNA binding stimulates the ATP hydrolysis of gp16. The stimulation extent was dependent on the structure and chemistry of DNA or RNA, strongly suggesting that gp16 interacts with pRNA as a part of ATP hydrolyzing complex in the packaging motor, and its ATPase activity can be stimulated via their interactions. To understand the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was determined as a function of DNA structure or chemistry. The kcat and Km in the absence of DNA was 0.016/s and 351.0 µM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of pRNA or DNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine) > dsDNA > poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single-molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involving in contact with gp16 during DNA packaging.
Keywords: phi29 DNA packaging motor, pRNA, packaging enzyme
Abstract:
Retroviral RNA encapsidation involves a recognition event between genomic RNA packaging signals and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) protein domain of Gag is known to be involved in genomic RNA packaging, and displays nucleic acid binding, aggregation, and chaperone functions. In contrast, HTLV-1 NC, a member of the deltaretrovirus genus, displays very weak nucleic acid binding affinity and very little chaperone or aggregation activity. It has also been demonstrated that mutation of conserved charged residues in the BLV matrix (MA) domain affects virus replication and viral RNA packaging efficiencies in vivo. Based on these observations, we hypothesized that the MA domain of Gag may generally contribute to nucleic acid binding and genome encapsidation for the deltaretroviruses. To gain further insight into the nucleic acid binding properties of MA, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various nucleic acid constructs in vitro. Mutagenesis studies were designed to probe the role of conserved charged amino acid residues of MA in aggregating and binding nucleic acids. Fluorescence anisotropy measurements were performed to measure nucleic acid binding affinity. In general, HTLV-2 MA displays substantially higher binding affinity to nucleic acids and better chaperone and aggregation activities than either HIV-1 MA or HTLV-2 NC. The simultaneous mutation of two basic residues (R47A/K51A) in HTLV-2 MA ¦Á-helix II, results in a binding defect to non-specific ssDNA relative to wild-type, whereas single point mutations have more modest effects on binding. HTLV-2 MA binds with higher affinity and apparent specificity to SL2 RNA derived from the putative packaging signal of HTLV-2. Furthermore, an HIV-1 MA triple mutant, E40R/E42L/N47K, designed to mimic HTLV-2 MA ¦Á-helix II, dramatically improves binding affinity and chaperone activity of the HIV-1 MA protein in vitro and restores RNA packaging to a ¦¤NC HIV-1 variant in vivo. Taken together, these results are consistent with a role for HTLV-2 MA in deltaretrovirus RNA packaging.
This work was supported by contract NO1-CO-12400 with SAIC-Frederick, Inc.
Keywords: deltaretrovirus, matrix protein, packaging
Abstract:
Many DNA or RNA-binding proteins form a hexameric ring, but the mechanism of hexamer assembly is poorly understood. The pRNA of bacteriophage phi29 DNA packaging motor also forms a hexameric ring. It is generally believed that the specificity in Protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching. Here we used single molecule studies to elucidate a mechanism suggesting that the specificity and affinity in Protein/RNA interaction relies on RNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form hexamer prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA falling off the motor. Extension or reduction of the ring circumference failed in motor-binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and drove the motor to package DNA and produce infectious virion phi29. Single molecule imaging confirmed that the copy number of pRNA on the motor is the common multiple of two and three, that is, a hexamer. This novel mechanism will be presented at this RNA meeting.
Keywords: phi29, pRNA, connector
Abstract:
Paranemic crossover (PX) motifs provide specific, programmable and reversible binding interactions between pre-folded nucleic acid molecules (RNA or DNA). They involve inter-molecular Watson-Crick basepairing with minimal disruption of the secondary structures of the interacting nucleic acids.1 Minimal paranemic motifs involve two strand crossovers and can be programmed across the major or minor grooves of the interacting molecules.
We will present applications of PX self-assembly to RNA biosensing and RNA nanotechnology. Sequence-specific, label-free RNA biosensors (“TokenRNA”) targeting prefolded internal loop motifs (“Target RNA”) were constructed by coupling paranemic binding motifs to a Malachite Green (MG) aptamer obtained by SELEX.2 MG fluorophore added to the solution, only fluoresces when bound to the RNA aptamer. The aptamer-containing “TokenRNA” only binds MG in the presence of its RNA target. We show that this binding is sequence-specific as single-basepair mismatches in the paranemic binding motif disrupt the TokenRNA-Target interaction.3 We also explored the use of the paranemic motif as a cohesion tool for engineering linear RNA fibrils. Atomic Force Microscopy (AFM) showed that linear fibrils exceeding 2 micro-meters were obtained. The fibrils can be derivatized for multiple nanotechnology applications.
References:
References:
1. K. Afonin, D. Cieply, and N. Leontis (2008) J. Am. Chem. Soc. 130: 93.
2. D. Grate and C. Wilson (1999) Proc. Natl. Acad. Sci. USA 96, 6131.
3. K. Afonin, E. Danilov, I. Novikova, and N. Leontis (2008) Chembiochem. 9, 1902.
Keywords: Paranemic Motif, TokenRNA, Biosensor
Abstract not available online - please check the printed booklet.
Abstract:
Multiple Sequence Alignments (MSAs) of both protein and nucleic-acid sequences are a ubiquitous method for modeling sequence motifs that pervade every biological domain. Despite their utility, MSAs and methods derived from them fail to capture interpositional relationships that can be as critical to family membership as are positional identities. This deficiency is particularly problematic when considering short nucleic-acid motifs such as endonuclease targeting signals or protein binding sites, since conservation of structure in these signals is often of equal or greater importance than conservation of nucleotide sequence.
The StickWRLD project has developed novel methods to quantitate as well as visualize interpositional relationships between residues that are signature features of some family alignments. We have identified dependencies in many protein and nucleic-acid families that are critical indicators of family membership. Some of these dependencies cannot be modeled by any existing family modeling method, including Hidden Markov Models. In some cases, the dependencies are sufficiently strong that Consensus, Position-Specific Scoring Matrix, and HMM methods all produce models that score sequences that are specificially excluded from the family, as more likely candidate members than any actual members of the family.
StickWRLD provides the researcher with a WWWeb-based interface to a visual survey of a family, enabling the researcher to determine if modeling the family in standard tools is appropriate. Ongoing research on the project is developing improved sequence modeling tools that incorporate interpositional dependencies into family models for sequence searches and classification.
References:
Hatice Gulcin Ozer and William C. Ray. �Informative Motifs in Protein Family Alignments�, Lecture Notes in Bioinformatics 4645, pp 161-170, 2007.
Hatice Gulcin Ozer and William C. Ray. �MAVL/StickWRLD: Analyzing Structural Constraints using Interpositional Dependencies in Biomolecular Sequence Alignments�, Nucleic Acids Research, vol 34, Web Server Issue, pp W133-W136, 2006.
William C. Ray. �MAVL/StickWRLD: Visualizing Protein Sequence Families to Detect Non-Consensus Features�, Nucleic Acids Research, vol 33, Web Server Issue, pp W315-W319, 2005.
Keywords: bioinformatics, alignment, structure
Abstract not available online - please check the printed booklet.
