Compositional changes within the spliceosome

Despite the indications that pre-mRNA splicing catalysis is at least partially RNA-based, the spliceosome, unlike group II introns, requires a plethora of protein factors to assemble the introns and the snRNAs in a catalytic structure. These proteins play crucial roles in SS recognition; facilitate dynamics of RNA-RNA and RNA-protein interactions and ensure the proper arrangement of the catalytic centers of the spliceosome.

The protein composition of affinity purified spliceosomal complexes stalled at certain assembly stages has been determined by mass spectrometry. These studies demonstrated that the composition of the spliceosome is highly dynamic with remarkable exchanges of proteins from the assembly stage, throughout activation and disassembly (Makarov et al., 2002;

Makarova et al., 2004; Behzadnia et al., 2007; Bessonov et al., 2008, 2010; Deckert et al., 2006).

Besides the human and D. melanogaster spliceosomes, the protein composition of affinity purified, in vitro assembled S. cerevisiae spliceosomal complexes have been recently determined (Fabrizio et al., 2009). It can be noted that the yeast splicing machinery contains the evolutionary conserved core of spliceosomal proteins required for constitutive splicing.

Additional proteins found in higher Eukarya spliceosomes are mainly implicated in alternative splicing, a process mostly absent in yeast.

A dramatic exchange of proteins occurs during spliceosome assembly and activation.

Interestingly, the proteins involved in dissociation/recruitment during B complex to C complex transitions are homologous in yeast and metazoans indicating that not only the proteins but also the compositional dynamics of the splicing machinery are evolutionarily conserved (Fabrizio et al., 2009; Bessonov et al., 2008, 2010).

Proteomic analysis of human spliceosomal A complex revealed that it consists of ten A complex-specific proteins besides U1 and U2 snRNPs (Behzadnia et al., 2007). These non-snRNPs leave the spliceosome during A to B complex transition while ~60 other proteins are recruited. Thus, the B complex contains U1 and U2 snRNPs, the U4/U6.U5 tri-snRNP plus 35 non-snRNP proteins, including the hPrp19/CDC5L and RES complexes and a group of B complex-specific proteins (Deckert et al., 2006). In the conversion from B to B^act complex, all U1 and U4/U6 proteins are lost. In contrast, several proteins are recruited to or become more stably associated with the spliceosome. All hPrp19/CDC5L complex proteins as well as related proteins are more abundant in B^act. The presence of hPrp2 in the purified B^act complexes indicates that these complexes have not yet undergone catalytic activation, as Prp2

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is known to dissociate from the spliceosome after catalyzing the remodeling step that yields a catalytically active B* complex (Kim and Lin, 1996). In the transition from B^act to C complex, the new additions to the spliceosome mainly consist of C-complex specific proteins and the so-called step II factors, which are proteins known to function prior to or during the second transesterification reaction. Additionally, SF2 helicases and peptidyl-prolyl isomerases (PPIases) are recruited to the C complex, potentially playing a role in RNP remodeling at this stage of splicing. Furthermore, SF3a and SF3b proteins are specifically destabilized from the human spliceosome during B^act to C complex transition. Finally, members of the exon junction complex (EJC), which are important for mRNA translation, are recruited at this stage (Bessonov et al., 2008, 2010).

In yeast, drastic rearrangements occur in the transition from B to B^act complex (Fig. 1.7), the latter representing the spliceosome prior to the final catalytic activation mediated by Prp2.

U1 snRNP is released as well as the U4 snRNA and all the U4/U6 associated proteins. At this stage, 12 B^act proteins are recruited that may be either involved in establishing/stabilizing U2/U6 base-pairing (such as Ecm2 and Cwc2) or promoting step 1 (Prp2, Spp2 and Yju2).

Comparison of B^act with C complexes (Fig. 1.7) shows that key proteins that are required at later stages of splicing are recruited during this transition. At least nine proteins, mainly step 2 factors, as well as the trimeric disassembly NTR complex join the spliceosome during C complex formation (Fabrizio et al., 2009). Due to the limited number of proteins recruited during B^act to C complex transition, it has been possible to investigate the requirements of some of these factors for step 1 or 2 by complementing purified spliceosomes (B^actprp2), which were stalled before the catalytic activation step mediated by Prp2, with purified recombinant splicing factors (Warkocki et al., 2009). The ability to rescue both steps of splicing in yeast using purified components could possibly help to elucidate the role of some RNA helicases implicated in RNP remodeling during activation.

It is important to mention that not only the protein composition during the splicing cycle changes but also the extent to which these proteins are modified. Several spliceosomal proteins are post-translationally modified prior to or during their involvement in the splicing cycle (Mathew et al., 2008; Soulard et al., 1993). Post-translational modification patterns constitute a code for recruitment of mRNA processing factors once they generate structural-mediated transitions that provide new interaction platforms. Consequently, these modifications play regulatory roles in the progression of splicing (Wahl et al., 2009).

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Figure 1.7: Compositional dynamics of the yeast spliceosome. The protein composition of B, B^act and C complexes of S. cerevisiae, identified by mass spectrometry, are shown. Proteins are grouped according to their function or association with a snRNP. The relative abundance of the proteins is indicated by the light (substoichiometric) or dark (stoichiometric) lettering. Considering that the compositional dynamics of the splicing machinery are evolutionarily conserved between yeast and human, only the representative scheme of the compositional dynamics of the yeast spliceosome is shown for simplicity.

Several enzymes responsible for introduction or removal of post-translational modifications are found in the spliceosomal complexes, such as SR protein kinases 1 and 2, Prp4 kinase and Clk/Sty kinase. In general, phosphorylation predominantly occurs during spliceosome assembly and activation, whereas dephosphorylation is more prominent during

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catalysis and disassembly. Furthermore, spliceosomal proteins seem to undergo other types of modifications such as ubiquitination (Bellare et al., 2008) and acetylation (Kuhn et al., 2009).