|dc.description.abstracteng||Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex. It assembles anew on each pre-mRNA intron by the stepwise binding of five snRNPs (U1, U2, U4, U5 and U6) and numerous proteins leading to the formation of the spliceosomal complex B which does not have yet an active catalytic site. For the establishment of the active site major structural changes are required, resulting in the formation of the activated B complex (Bact), which is then converted into the catalytically activated B* complex by the action of the Prp2 RNA helicase. Following the recruitment of the splicing factor Cwc25, the first step of splicing occurs, whereby the 5' splice site of the pre mRNA is cleaved and the 5' end of the intron is ligated to the branch site adenosine to form a lariat-like structure; concomitantly the C complex is formed. At this time the second step of splicing occurs which leads to exon ligation. The newly formed mRNP is released from the spliceosome and the intron lariat spliceosome is disassembled. The released snRNPs are thought to re-assemble for a new round of splicing.
A complex RNA–RNA network involving the snRNAs and the pre mRNA is formed during spliceosome assembly, and the resulting RNA structure plays a central role in catalysing the two steps of splicing. During spliceosome activation, U6 snRNA rearranges and forms an internal stem-loop (ISL) which plays a central role in the catalysis of splicing. The U6-ISL contains an internal bulge region that is critical for metal-ion binding and contains functionally important residues. U6 snRNA also forms base pairs with U2 snRNA generating the U2/U6 helix I. Finally, U6 snRNA via its conserved ACAGAGA sequence, also forms base pairs with the 5' end of the intron. In this arrangement, the branch site is juxtaposed with the 5' splice site.
While the importance of individual RNA-structural elements such as U6 ISL, U2/U6 helix I and the U6 ACAGAGA/5' splice site helix for splicing catalysis is well established, little was known at the time I started this work about how these various RNA elements are brought into a catalytically active tertiary conformation. Interestingly, if one examines how the catalytic center of the group II self-splicing introns is organized, a number of similarities between pre-mRNA and group II intron splicing can be recognized and indicate that the RNA elements of the respective catalytic core adopt similar folds in both systems. These include (i) the identical chemistry of the catalytic steps of both kinds of splicing and (ii) the great similarity between catalytically important structural elements in group II introns and the spliceosomal RNA network, especially between domain V (DV) of group II introns (which forms a stem-loop) and the U6 ISL, both of which bind catalytically active metal ions. One of the most impressive features revealed by the recently published crystal structure of an intact self-spliced group IIC intron is how numerous long-distance interactions between conserved structural elements of DI to VI and DV are essential to induce an unusual, catalytically important fold in DV.
In view of the paucity of conserved RNA tertiary structures in spliceosomal introns that might direct the folding and juxtaposition of essential catalytic RNA-structural elements (i.e U6 ISL and U2/U6 helix I) into an active conformation, it seems likely that spliceosomal proteins may have taken over this function, at least in part. Good candidates would be one or more of those proteins that become stably integrated into the spliceosome during its activation (i.e., the formation of the Bact complex). In yeast, these include a protein complex termed the “nineteen complex” (NTC) that consists of eight core proteins, and an additional set of NTC-related proteins. Among these, the yeast Cwc2 protein was of particular interest since it has an RRM and a zinc-finger domain, is essential for pre mRNA splicing in vivo and has been shown to contact U6 snRNA during splicing in yeast extracts.
Here we show that Cwc2 is essential for the first step of splicing in vitro, and that it is not required for the Prp2-mediated remodelling step that generates the catalytically competent B* complex. We demonstrate that in purified catalytically active spliceosomes, Cwc2 contacts the U6 ISL, as well as regions of the U6 snRNA and the intron adjacent to the 5' splice site. Chemical structure-probing further suggests that Cwc2 may also directly or indirectly contact U6/U2 helix I. Thus, our data place Cwc2 at the heart of the spliceosome's catalytic center. During this time the crystal structure of the Cwc2 functional core was solved by our group, and could be used to determine structure-function relationships by rational mutagenesis of Cwc2 combined with splicing. In addition, mass spectrometric analysis of RNA-protein crosslinks and electrophoretic mobility shift assays (EMSA) showed that Cwc2 acts as a multipartite RNA binding platform to bring RNA elements of the spliceosome’s catalytic center into an active conformation.
Interestingly, we also show that RNA interactions involving Cwc2 are evolutionarily conserved, as demonstrated by studies of its human counterpart RBM22, indicating that the observed Cwc2/RBM22 RNA contacts in the spliceosome are functionally important. We propose that Cwc2, in co operation with the essential splicing factor Prp8, induces an active conformation of the catalytic RNA elements in the spliceosome. In conclusion, our data suggest that the function of RNA-RNA tertiary interactions within group II introns, that is, to induce a catalytically active RNA conformation of DV, has probably been taken over by proteins that contact the functionally analogous U6-ISL, within the spliceosome.||de