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Pre-mRNA splicing is catalyzed by the spliceosome in a two-step reaction. Both catalytic steps have now been reconstituted using purified, defined components. This system identifies a role for Cwc25 in the first step of splicing and allows future detailed mechanistic analyses of splicing. The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 455 activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Small nuclear ribonucleoprotein (snRNP) particles are essential for pre-messenger RNA splicing. In human HeLa cells, 40 proteins associated with snRNPs have been identified. Yet, the function of many of these proteins remains unknown. Here, the immunoaffinity purification of the spliceosomal snRNPs U1, U2, U4/U6.U5, and several nucleolar snRNP species from the yeast Saccharomyces cerevisiae is presented. The U1 and U4/U6.U5 snRNPs were purified extensively and their protein composition and ultrastructure analyzed. The yeast U1 snRNP is larger and contains three times more specific proteins than its human counterpart. In contrast, the size, protein composition, and morphology of the yeast and the human U4/U6.U5 snRNPs are significantly similar. The preparative isolation of yeast snRNPs will allow the cloning as well as genetic and phylogenetic analysis of snRNP proteins which will accelerate our understanding of their function.

The yeast U4/U6.U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6.U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6.U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6.U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

The yeast U4/U6⋅U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism. pre-mRNA splicing mass spectrometry

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4+U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.