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Maquat and Yu Labs. The Maquat lab studies a pathway that typifies more than one-third of all genetic and acquired diseases, including cancer. This pathway, called nonsense-mediated mRNA decay (NMD), eliminates mRNAs that prematurely terminate translation and, thus, have the potential to encode deleterious truncated proteins. Maquat-lab studies of NMD have revealed links between pre-mRNA splicing in the nucleus and mRNA translation in the cytoplasm as well as a new template for protein synthesis, called the “pioneer” translation initiation complex, and insight into new aspects of mRNA-protein interactions. An understanding of the molecular biology of RNA metabolism and, in particular, the NMD pathway is key to devising intelligent therapeutics to treat the many nonsense-associated diseases. New reagents that promote nonsense suppression and, thus, the production of full-length functional proteins from disease-associated genes are currently under development in work spearheaded by the Yu lab.

Robert Bambara, Baek Kim and Lisa Demeter. HIV-1 uses frequent recombination between its two RNA genomes to create viral diversity. This diversity helps the virus to escape host immune response and drug therapy. Recombination can occur by strand transfer between RNA templates. This mechanism is also employed for the minus-strand strong stop transfer in the replication pathway. Transfer involves a shift of the growing cDNA primer from the original donor RNA to a second acceptor RNA. Our work reconstituting recombination in vitro with pure proteins, and in vivo in cell culture, addresses mechanisms that drive strand transfer. Transfers initiate at sites where the virally encoded reverse transcriptase (RT) pauses, allowing it to use its RNase H function to concentrate cuts in the donor template. Transfers can occur by a multistep process in which acceptor template invades the DNA at the gapped site in the donor template. The cDNA-acceptor hybrid spreads until the 3’ terminal region of the cDNA completes transfer. However, numerous questions remain. Minus-strand transfer model reactions in vitro indicate that RNA folding is an important determinant of transfer efficiency. We are investigating how folding determines whether the cDNA end can be inactivated before transfer occurs. New results indicate that transfers can occur by a mechanism called proximity, which does not involve spreading of the initial hybrid. We will evaluate the relative contributions of spreading versus proximity mechanisms. Evidence suggests that RT is obligated to dissociate for transfers, and that RT must exercise its unique 5’ end-directed RNase H activity. We are determining whether either or both functions are essential for transfer. Lastly, we developed a viral-cell culture system capable of measuring the positions and frequencies of recombination crossovers over more than half of the length of HIV-1 at a resolution of 25 nucleotides. We focused our initial sequencing on a 459-bp region spanning from DIS in the gag gene. It revealed a striking peak of recombination in which two-thirds of crossovers in the region occurred in a span of about 100 nucleotides. Significantly, we successfully recapitulated the hot spot in strand transfer assays in vitro. This will allow us to determine its structural and mechanistic basis. Overall, results of our work will clarify the exact mechanisms and requirements of strand transfer in HIV-1. This is a first step to therapeutic targeting of strand transfer as a means of interfering with HIV-1 infection.

Mathews, Sharma, Turner labs. To understand RNA function, knowledge of structure is required. We collaborate on improving methods for predicting RNA secondary structures --  the set of the AU, GC, and GU base pairs --  from sequence. The Turner lab studies the stability of model RNA structures, as measured by free energy change, in order to improve the set of rules for predicting the sequence-specific stability. These sequence-dependent rules for predicting free energy change are the basis for the prediction of structure by free energy minimization using the computer program, RNAstructure. The Mathews and Sharma labs work together to improve algorithms for predicting structures shared by homologous sequences.  The Mathews lab is applying these algorithms to discover novel RNA genes in the human genome that relate to human health and disease.