Structural Studies of the Yeast Prp8-Snu114 Complex

Abstract

The spliceosome catalyzes the removal of non-protein-coding introns and ligates neighbouring exons in pre-mRNAs with single nucleotide precision to produce mature mRNAs. Prp8 and Snu114 are highly conserved proteins that form a stable heterodimer as part of the U5 snRNP. They play essential roles in the regulation of the spliceosome and the formation of the catalytic core for the two transesterification reactions. Prp8 is located at the heart of the spliceosome, and considered the “master regulator” of spliceosomal activity. It contacts all the crucial pre-mRNA and snRNA elements involved in splicing: 5' SS, BP, 3' SS, U2, U5 and U6, and genetic interactions have been detected between PRP8 and numerous components of the spliceosome. Prp8 forms a large cavity that is proposed to accommodate the catalytic centre of the spliceosome. Snu114 is a GTPase homologous to ribosomal translocases EF-G and eEF-2. Both Prp8 and Snu114 modulate the activity of Brr2, a DExD/H RNA helicase. Brr2 is required for the ATP-dependent unwinding of the U4/U6 snRNA during spliceosome activation, as well as spliceosome disassembly after the release of the mRNA product.Recent technological developments have enabled determination of 3D reconstructions at near atomic resolution of a broad range of biological samples by single particle electron cryo-microscopy (cryoEM). The single-electron counting K2 Summit camera gives noiseless readout, and its high frame rate enables recording image with a novel dose fractionation (or movie) procedure which can be coupled with a newly developed motion correction algorithm to correct beam induced image blurring. I am purifying the Prp8-Snu114 complex from S. cerevisiae, and will attempt to use the latest high resolution cryoEM technique to solve the its structure.