Structural and dynamics studies of post-transcriptionally modified snRNAs

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Project Summary: There is increasing evidence that post-transcriptional modifications play essential roles in the biological functions of coding and non-coding RNAs. More than 100 chemically-modified RNA nucleosides have been identified to date that can impact RNA fate and function. 2'-O-methylation (Nm) of the 2'-OH position is a unique modification that impacts the ribose sugar moiety of all four nucleosides. Nm is found in high abundance in ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). It is also present on microRNA, at the 5'-cap of messenger RNA (mRNA), and recently discovered internally on mRNA. Nm is critical for the proper functioning of many of the above RNAs, and in several cases, loss of Nm has been linked to clinical conditions. Despite its importance, how Nm affects RNA cellular activity remains poorly understood at the molecular level for the majority of these RNAs. The cellular functions of many regulatory RNAs rely on finely-tuned changes in RNA structural dynamics that take place in response to specific cellular cues such as the binding proteins, ligands, or other RNAs. A prominent example is the spliceosome machinery, which catalyzes mRNA maturation. RNA dynamics plays essential roles in the assembly and disassembly of the spliceosome as well as in cycling between the different conformational states required for catalysis. U2-U6 and U4-U6 snRNA complexes are critical dynamic structural elements of the spliceosome, which are highly enriched in Nm modifications. The role of these modifications on snRNAs remains poorly understood. This project will determine how Nm modifications influence snRNA structure and splicing. Aims 1 and 2 will utilize advanced Nuclear Magnetic Resonance (NMR) techniques, including Relaxation Dispersion experiments (RD), and additional biophysical techniques to test the hypothesis that loss of Nm modifications affects the stability, hybridization kinetics, and conformational dynamics of snRNA structures. Aim 3 will use siRNA-mediated knockdown and genetically-defined knockout cell lines to determine which snRNA modifications have the greatest impact on mRNA splicing in cardiac cells. The structural dynamics studies of Aims 1 and 2 will therefore complement the functional studies of Aim 3. Together, these studies will significantly expand our knowledge of how Nm modifications contribute to activity (for instance splicing of cardiac genes) via altering the dynamic and structural properties of snRNAs.