ZIA BC 008382 (ZIA) | |||
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Title | Computational Approaches for RNA Structure/Function Determination | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Shapiro, Bruce | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $245,365 | Project Dates | 10/01/1983 - 00/00/0000 |
Fiscal Year | 2014 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Digestive Diseases (10.0%) |
Brain (10.0%) Liver Cancer (10.0%) Nervous System (10.0%) |
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Research Type | |||
Development and Characterization of Model Systems Application of Model Systems |
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Abstract | |||
Our previous discovery of the turnip crinkle virus tRNA-like translational enhancer (TCV TSS) motivated us to search for similar elements in other viruses. As a result, the middle of the UTR of the pea enation mosaic virus (PEMV) was found, by our modeling, to have a comparable T-shaped 3D motif (kl-TSS), a CITE, that is similar in both structure and function to the TCV element. This structure, however, does not contain pseudoknots like the TCV element. It was shown that a hairpin loop in the T-shaped PEMV engages in a long-distance kissing loop interaction with a hairpin stem loop just down-stream of the start codon. It was also shown that ribosomes bind to the kissing complex as well as to the T-shaped element alone. Analysis indicated that mutations in the element that reduced translation also reduced ribosome binding or the RNA-RNA kissing loop kl-TSS interaction. This element is also presumed to be important not only for delivery of ribosomes to the 5' end of the virus, but also in associating the eukaryotic initiation factor eIF4E with the 5' end due the binding of eIF4E with a nearby downstream element the PTE. We developed the programs CovaRna and CovStat to explore long-range co-varying RNA interaction networks using whole genome alignments. This new methodology was applied to Drosophila genomes. A parallel version of the program was devised to speed-up processing and the algorithms also rely on fast indexing schemes and conservative statistical methods to determine highly significant interactions. The methodology found interesting interactions that appear to be related to endogenous siRNAs, gene transport, and to synchronized mutations that may include common interaction partners. These results indicate the potential importance of such networks. The World Health Organization estimates there may be as many as 50-100 million cases of dengue fever per year. We characterized the untranslated regions of the virus focusing on the 3' UTR and its effects on replication and translation. We used MPGAfold and StructureLab to analyze the folding characteristics of a subgenomic (minigenome) RNA from dengue type 2 as well as mutated variants. We previously showed that MPGAfold is capable of predicting the secondary structure and folding dynamics including co-transcriptional folding of various RNAs. Our computational results were correlated with experiments on a 719 nt minigenome consisting mostly of the 5' and 3' UTRs and a dengue type 2 replicon containing a Renilla luciferase reporter. Attention was focused on the formation of two dumbbell-like structures found to form in the 3' UTR with the putative formation of pseudoknots involving regions downstream from the dumbbells. The computational results showed a significant preference for the formation of the 3' dumbbell over the 5' dumbbell. Mutational studies indicated that the motifs involved in dumbbell formation including the putative pseudoknots were important for replication but had less significant varying effects on translation. In a later related study Selective 2'-hydroxyl Acylation Analyzed by Primer Extension (SHAPE) analysis was done on the minigenome and the mutational variants. Most of the MPGAfold predicted structures were found to be present by SHAPE, including the 5'-3' interactions and the dumbbell structures. SHAPE found evidence for the formation of the 5' pseudoknot structure, but the formation of the 3' pseudoknot appeared to be complicated by the presence of a portion of the 3' pseudoknot sequence being involved in the cyclization motive involving a 5' sequence. Understanding sequence related conformational characteristics of kissing loop formation is important for understanding the formation of these motifs in RNA viruses such as HIV and is also important for understanding their formation for use in RNA-based nanodesign. We explored two HIV-1 kissing loop monomers (from subtype-A and subtype B). Our hypothesis in this study was that the structure of the mono" |