Although many spliceosomal components are known, the interaction dynamics between them and the role the sub-complexes play in primary transcripts splicing regulation remain to be determined 
. For the yeast S. cerevisiae
, it is known that interactions between snRNAs and specific proteins can enhance or repress splicing activity and/or regulate spliceosome activation under different environmental conditions or developmental stages 
. It is possible that specific components and its interactions with the primary transcripts facilitate and control this process. We have previously shown that the depletion of Cwc24p affects splicing of pre-U3 snoRNA in vivo
. However, one of the greatest challenges that emerged from that work was to understand whether Cwc24p associates and interferes with pre-U3 spliceosomes. In this work, we performed in vitro
splicing experiments with Cwc24-immunodepleted extracts, using different substrates. Our data showed that this 30 kDa protein is a general splicing factor required for the stable association of U2 snRNP with pre-RNAs and thus important for spliceosome assembly, especially of those primary transcripts containing non-canonical branchpoint sequences.
In accordance with the hypothesis of Cwc24p being a general splicing factor, depletion of Cwc24p in in vitro
splicing reactions led to a reduction of the mature RNAs formed from the three different substrates used, pre-U3, pre-TEF4, and pre-ACT1. Splicing of all three transcripts tested was inhibited upon depletion of Cwc24p. Consistently with previous microarray data, U3 was very strongly affected. Interestingly, both U3 and snR38 (the snoRNA found in TEF4 intron) are box C/D snoRNAs. This might indicate a connection between the splicing and box C/D snoRNP assembly machineries. The great majority of mammalian snoRNAs are coded in introns and, consistently, snoRNP assembly is dependent on interactions between components of the spliceosome and the snoRNP machineries. IBP160 was isolated as one of such proteins, connecting splicing of primary transcripts containing snoRNAs in the introns to proteins of snoRNP complex 
. However, in S. cerevisiae
, only 7 snoRNAs are coded within introns, 6 of which are snoRNAs of box C/D 
. The splicing defects observed upon Cwc24p depletion along with its interactions with components of splicing machinery (Cef1p and Brr2p) and snoRNP assembly (Nop17p) 
, could indicate that this protein mediates these processes. In accordance with that, the addition of recombinant Cwc24p restored the efficiency of splicing of pre-U3 snoRNA, confirming that the splicing defect is specifically due to the depletion of Cwc24p. Taken together, the splicing defects observed under Cwc24p depletion indicate this is a general splicing factor, but especially required for pre-U3 splicing.
In S. cerevisiae
, the U3 snoRNA is transcribed from two different genes, snR17A and B, which code for pre-snoRNAs containing 157-nt or 130-nt introns, respectively. Despite its canonical 5′ and 3′ splice sites, the branchpoint sequence is not conserved in this pre-snoRNA, with a G instead of a U in the first position. The mature U3 snoRNA is essential for the initial cleavages of the ribosomal RNA precursor 
and, consistently, depletion of Cwc24p also affects pre-rRNA processing 
. In fact, our data supports the hypothesis that splicing of this transcript, that has a non-consensus branchpoint sequence, depends on Cwc24p. Yeast splicing is strictly regulated and consensus sequences are essential to guide the spliceosome 
, through interactions with its components and with the precursor RNA. In S. cerevisiae
, in addition to pre-U3 snoRNA (snR17A and snR17B), twelve other transcripts have this non-canonical branchpoint site (Ares Lab Yeast Intron Database, http://intron.ucsc.edu/yeast4.1/
). We also tested in vivo
splicing on two such transcripts, IWR1 and YRA1, and showed that Cwc24p depletion severely affected splicing of both. These results confirm the hypothesis of an important role for Cwc24p on splicing of primary transcripts with non-canonical branchpoint sites.
During spliceosome assembly, the branchpoint site on the precursor RNA is important to recruit and stabilize the U2 snRNP. Several rearrangements on the U2 snRNP particle and its pairing with U6 snRNA are important for catalytic activation and their positioning on the primary transcript 
. U2 snRNA pairing with the branchpoint site also prevents the reaction from happening until rearrangements promoted by Prp2p allow the transesterification reaction to occur 
. In this sense, a non-consensus branchpoint might be weakly bound by U2 snRNP, destabilizing the complex and leading to inefficient spliceosome activation. Indeed, our data shows that Cwc24p depletion causes a strong reduction on U2 and U6 association with pre-U3A spliceosomes, which could be due to the fact that either U2 or the pair U2/U6 is unstable in these complexes. Interestingly, Cwc24-depleted reactions using a 3′ splice site mutant substrate (ACAC pre-U3), which blocks spliceosome progression after the first step of splicing, caused a reduction in U2 levels but did not alter U6 levels bound to the complexes. This suggested that the depletion of Cwc24p destabilized the association of U2 snRNA, but did not affect the U6 snRNA recruitment. It is possible that Cwc24p facilitates either recruitment or stabilization of U2 snRNP on the pre-U3 substrate. However, our data showing the importance of Cwc24p for snRNAs precipitation with TAP-Prp19 spliceosomes, along with previous data showing the enrichment of Cwc24p on Bact
, and its association with NTC complex 
favor the latter hypothesis. Therefore, Cwc24p may be important for the stabilization of U2 snRNP, helping to promote the rearrangements necessary for the catalytic activation.
This suggests an important role for Cwc24p on spliceosome assembly, which ultimately leads to efficient splicing. It is also important to consider that U2 association and stabilization on the spliceosome is highly dependent on U2 snRNP proteins, for example the SF3a/b proteins 
. Although we were unable to detect an interaction between these proteins and Cwc24p, it is possible that transient interactions occur. Interestingly, recent high-throughput analysis with the human orthologue of Cwc24p (RNF113) showed that this protein interacts with subunits of the Bact
complex in the yeast two-hybrid system 
. Weak splice sites have been shown to be subjected to specific regulation by protein components of the spliceosome. Hub1p facilitate splicing of transcripts with a deficient 5′ splice site 
. The association of MER2 transcript weak 5′ splice site with U1, for example, is dependent on the interaction between Mer1p and U1 snRNP proteins during meiosis 
. Mer1p binds to an intronic enhancer and mediates U1 snRNP association leading to spliceosome assembly on this substrate 
The spliceosome is a multi-megadalton machinery composed of ~60 protein components but more than 100 proteins participate in its assembly 
. Cwc24p was isolated on a multi-protein complex in association with Cef1p 
. Proteomic analysis showed that, similarly to Prp19p and Cef1p, Cwc24p is already present in B complex, with enrichment in Bact
complex just prior to activation. But, also similarly to most of other Cwc proteins, fewer Cwc24p peptides were detected in C complex, indicating that its association with the catalytically active complex is less stable 
. Prp19p and Cef1p are major subunits of the NTC complex, and along with other components, promote several rearrangements important for catalytic activation 
. Cwc24p physically interacts with Cef1p 
, and our results showed that it is required for Prp19p association with the spliceosome. In the absence of Cwc24p, either because the NTC is incomplete, or because some essential interactions with other splicing factors are missing, the binding of Prp19p to the spliceosome is compromised. Regarding Cwc24p role in U2 binding to the primary transcripts, it is possible that Cwc24p acts as a regulatory protein that indicates when the spliceosome is ready to perform catalysis. In addition, interaction with Brr2p might be important for positioning and stabilizing Cwc24p on the catalytic core of the spliceosome. In this sense, other Cwc proteins have shown strong effects on splicing regulation. Cwc21p and Cwc25p are important for rearrangements prior to the first step reaction, regulating the spliceosome activation 
In summary, our data shows that Cwc24p affects splicing in general by facilitating the stabilization of U2 on primary transcripts spliceosomes. This stabilization is especially important in the case of primary transcripts containing non-consensus branchpoint sequences. During the final preparation of this article a study was published on the Prp2-mediated proteins rearrangements at the catalytic core of the spliceosome 
. In that study it is reported that Cwc24p is stably bound to Bact
complex, but dissociates from it upon catalytic activation by Prp2p during transition to B*. That work complements the data reported here and confirms an early role for Cwc24p during splicing. In addition, Cwc21p, another protein associated with the NTC complex, was recently shown to be involved in the stabilization of the HRB1 pre-mRNA (which has a non-consensus branchpoint sequence) in the spliceosome catalytic center (Gautam, A., Grainger, R., Barrass, D. and Beggs, J.D.; personal communication), further suggesting the importance of the NTC complex for stabilizing the spliceosome on primary transcripts.