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 | $278,200 | Project Dates | 01/01/1983 - 00/00/0000 |
Fiscal Year | 2015 | 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. In a recent study we showed the existence of yet another T-shaped element in PEMV located near the 3' terminus that is similar to the TCV element containing 2 pseudoknots. This element also binds ribosomes. In addition, just upstream of this element there is a hairpin loop that interacts with the 5' end of the virus. This element was also shown to be important for translation. Programmed -1 ribosomal frameshift (-1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. We described and modeled a -1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated -1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA-miRNA interaction suggests that formation of a triplex RNA structure stimulates -1 PRF. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional -1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells. Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with significant clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. In this study we identified interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We found that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identifed premRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAFmelanoma. 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 computational prediction of the secondary structures within the 5' and 3' untranslated regions of the dengue virus serotype 2 (DENV2). For secondary structure prediction purposes we used a 719 nt-long subgenomic RNA construct from the DENV2, which we refer to as the minigenome. This minigenome has been shown to contain the elements needed for translation, as well as negative strand RNA synthesis. We showed how MPGAfold and StructureLab were used to analyze the folding characteristics of the minigenome. The computational results showed a signi |