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 significant preference for the formation of the 3' dumbbell over the 5' dumbbell, in addition to the UAR motif. However, the 5' dumbell motif appeared often as a transient motif. Co-transcriptional folding simulation also gave similar results that were consistent with experimental data. MicroRNA 137 has been implicated with other microRNAs to be associated with neurogenesis and brain tumor development. Specifically, its up-regulation has been shown to be important for neuro cell differentiation and down-regulation has been implicated in disease processes. A genome-wide target mapping was performed on glioblastoma cells. 1468 genes were found to be negatively impacted by miR-137. Computational analysis also revealed that many of the genes had a highly likely interaction with miR-137 seed region. Several of the targeted genes were related to neurogenesis and oncogenic proteins. Other miRNAs, namely miR-124, 128 and 7 also regulate targets that are regulated by miR-137 and are associated with neurogenesis and tumorgenesis, thus their increase or decrease can have significant implications for the genetic control of neurogenesis or tumorgenesis. Over 150 million people are infected with hepatitis C virus (HCV). We investigated the synergistic effects that infection by the virus has on the course of the virus. The elevation of the expression of IFNL3 mRNA is known to help in clearance of the virus. In particular, a new means was discovered whereby HCV affects this antiviral response. This mechanism is dependent on a mutation found in the 3' UTR of IFNL3 mRNA that controls transcript stability. The mutation had an effect on AU-rich dependent decay of the transcript and the binding of a microRNA induced by HCV infection. RNA structural analysis showed that the indicated mutation in the 3' UTR of IFNL3 had the potential to alter the structure and thereby prevent AU rich element (ARE) binding proteins from degrading the RNA. In addition, it was found that the mutation prevents the binding of microRNAs that cause down-regulation of this gene. RNA pseudoknots play important roles in many biological processes. Previous methods for comparative pseudoknot analysis mainly focus on simultaneous folding and alignment of RNA sequences. Little work has been done to align two known RNA secondary structures with pseudoknots taking into account both sequence and structure information of the two RNAs. This research involves the development of a novel method for aligning two known RNA secondary structures with pseudoknots. A partition function methodology is used to calculate the posterior log-odds scores of the alignments between bases or base pairs of the two RNAs with a dynamic programming algorithm. The posterior log-odds scores are then used to calculate the expected accuracy of an alignment between the RNAs. The goal is to find an optimal alignment with the maximum expected accuracy. A heuristic is developed to achieve this goal. The performance of the method was investigated and compared with existing tools for RNA structure alignment. The method has been implemented in a tool named RKalign, which is freely accessible on the Internet.
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