Project Summary The proposed research is focused on applying biophysical methods to understand the structure and function of RNA-protein complexes that regulate gene expression in eukaryotic cells. Our investigations will elucidate unexplored steps in the spliceosome assembly pathway, mRNA decay and RNA silencing. We propose to determine the structure of the U6 small nuclear ribonucleoprotein (U6 snRNP) particle and to describe how it associates with the U4 snRNP to form the U4/U6 di-snRNP. These interactions are critical for assembly and recycling of the spliceosome but are not yet understood at the molecular level. We will also investigate the Lsm1- 7 complex, which is highly related to U6 snRNP proteins but directs mRNA decay, a major post-transcriptional determinant of steady-state levels of gene expression. Our recent data suggest the Lsm1-7 complex is remodeled by its cofactor Pat1 in order to bind to a broad range of mRNAs. Finally, we will explore a newly discovered area of RNA biology involving the enzymatic addition of poly-UG (pUG) tails to RNA 3¢ ends. pUG- tailed RNAs are potent agents of gene silencing and establish the molecular memories required for trans- generational epigenetic inheritance in nematodes. We have discovered that pUG RNAs fold into an unusual quadruplex structure that explains the length requirement for RNA silencing in vivo. Humans have over a thousand internal pUG sequences within neuronal introns, which serve to regulate alternative pre-mRNA splicing through interactions with the protein TDP-43. We have discovered that pUG RNAs maintain their quadruplex structure when bound to TDP-43, the latter of which is involved in phase transitions and neurodegenerative disease. We will investigate the structural basis for this interaction and will collaboratively test our hypothesis in animals. By elucidating how pUG RNAs associate with proteins and small molecules we will better understand how these interactions direct distinct biological outcomes in diverse pathways such as RNA silencing and alternative splicing.