Structural analysis of the assembly machinery of spliceosomal U snRNPs

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The spliceosome is a macromolecular machine that catalyzes splicing of eukaryotic pre-mRNA. Four uridyl-rich small nuclear ribonucleoproteins (U snRNPs) are major building blocks of spliceosomes with various functions including identification of intronic sequences and the formation of its catalytic center. Each U snRNP contains a specific set of proteins dedicated to its specific task in splicing. However, they also have a set of seven Sm proteins in common that form the structural core of snRNPs. The formation of this Sm core, although spontaneously in vitro, follows a highly regulated and assisted pathway in vivo. It starts with the nuclear export of newly synthesized snRNAs and their assembly with Sm proteins in the cytoplasm. U snRNPs that have formed the Sm core translocate to the nucleus where additional particle-specific proteins are added. The mature particles are eventually incorporated into the spliceosome to enable splicing. Several labs including ours have identified a sophisticated machinery that assists the assembly of splicesomal U snRNPs in vivo. This machinery consists of at least 12 proteins that are organized in two cooperating units termed SMN-complex and PRMT5-complex, respectively. While the SMN-complex acts as RNP assembler and loads Sm proteins onto the snRNA, the PRMT5 complex functions upstream in this pathway. Its main task is committed by the assembly chaperone pICln, which forces Sm proteins into higher order structures (termed 6S complex and pICln-D3/B) required for the subsequent transfer onto the SMN-complex. In the past funding period, we have solved the atomic structure of two key intermediates of the snRNP assembly machinery. The 6S complex structure identifies the PRMT5 complex subunit pICln as an assembly chaperone and Sm-protein mimic, which enables the topological organization of five Sm proteins into a closed ring. A second structure of 6S bound to the SMN-complex components SMN and Gemin2 uncovers important mechanistic aspects of pICln elimination and Sm protein activation for snRNA binding. In this grant application, we wish to extend our studies on the assembly machinery focusing on the structural analysis of the entire SMN-complex and its dynamics during snRNP formation. Furthermore, the structural basis of 6S-formation on the PRMT5 complex will be investigated. From these studies we expect detailed structural and functional insight into critical steps of snRNP formation in vivo.