Polyadenylated nuclear (PAN) RNA is the most abundant lytic-phase transcript produced by the Kaposi's sarcoma-associated herpesvirus. Its high nuclear accumulation localizes to an expression and nuclear retention element (ENE). The U-rich internal loop of the PAN ENE sequesters the 32-poly(A) tail of PAN RNA into a triple helix (denoted as ENE+A) that protects the transcript from a novel rapid deadenylation-dependent nuclear RNA decay pathway. Similar ENE structures are formed by the 32-end sequences of two cellular long noncoding RNAs (lncRNAs): metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia beta (MEN?). These lncRNAs are highly abundant in cancer cells despite lacking canonical poly(A) tails. Instead, 32-genomically-encoded A-rich tracts are sequestered by the ENEs of MALAT1 and MEN? RNAs into proposed blunt-ended, triplexes. Because 3'->5'decay is typically initiated by an exonuclease binding to single-stranded RNA, the blunt-ends of cellular ENEs+A are likely subjected to different decay processes than the 32-poly(A) tails of viral ENEs+A. Moreover, the putative triplexes of cellular ENEs+A contain U?A-U triples interrupted by G and C nucleotides that are absent in all viral ENEs. I hypothesize that the different viral and cellular ENE+A structures are degraded via distinct decay mechanisms. The molecular basis for how viral and cellular ENE+A structures differentially block nuclear RNA decay has not been investigated. The enzymatic components of the rapid nuclear RNA decay pathway are unknown for PAN RNA and MALAT1 except that PAN RNA is likely deadenylated by poly(A)-specific ribonuclease (PARN).
In Aim 1, a series of cell-based assays will characterize the MALAT1 ENE+A in nuclear RNA decay, including time-resolved 32-RACE and RNA-based knockdown approaches to identify decay machinery.
In Aim 2, kinetic assays will establish the mechanistic basis for how viral and cellular ENEs+A inhibit decay enzymes during the binding and nucleotide excision steps.
In Aim 3, X-ray crystallography will be used to solve the structures of decay enzymes in complex with the PAN or MALAT1 ENE+A. Solving these structures will provide the first insights into how enzymes recognize and interact with triplexes. Initial kinetic and structural studies will use PARN and PAN ENE+A as a model system. Similar analyses will be performed with the MALAT1 ENE+A and its decay machinery uncovered in Aim 1. These experiments will elucidate the molecular mechanisms of how triple-helical RNA stabilization elements impede nuclear RNA decay while providing significant insights into the understudied pathways of nuclear lncRNA decay, the biological roles of RNA triplexes, and the mechanisms that stabilize MALAT1, a cancer-promoting lncRNA. This Career Development Award will allow me to develop my professional skills (e.g. classroom teaching) and to acquire specific technical training (e.g. X-ray crystallography) in the laboratories of Drs. Joan Steitz and Thomas Steitz at Yale University. These activities will enable me to secure an academic professorship and to build a top-tier research group.
This research project will provide insights into how specific RNA structures block destruction, allowing the RNA to accumulate to high levels in normal and cancer cells. Understanding these mechanisms will facilitate the design and development of novel therapeutics that prevent the accumulation of specific RNA molecules, specifically those contributing to cancer.