In this study, we show that Cwc22 is essential for pre-mRNA splicing both in vivo
and in vitro
. Although Cwc22 was previously reported to associate with the NTC component Cef1/Ntc85 (28
), we found that the association of Cwc22 with the NTC was very weak, as only tiny amounts of known NTC components were coprecipitated with Cwc22. Our data thus suggest that Cwc22 is not an integral component of the NTC. Furthermore, we demonstrate that Cwc22 has a function different from that of the NTC. It is not required for NTC-mediated spliceosome activation but is required for the first catalytic step in promoting the Prp2-mediated release of SF3a/b.
The catalytic steps of splicing involve more protein factors than expected. Each one proceeds through an ATP-dependent reaction followed by an ATP-independent reaction, and each requires a DExD/H-box RNA helicase, Prp2 and Prp16, respectively, for the ATP-dependent function. The ATPase activity of Prp2 is required for the release of SF3a/b (21
), presumably to clear the branch site for the binding of other factors to promote the first transesterification, which does not require ATP but requires Yju2, Cwc25, and HP-X (12
). In the second step, Prp16 mediates an ATP-dependent structural change of the spliceosome, resulting in the protection of the 3′ splice site (33
). Prp22, Prp18, and Slu7 are then required to promote the second transesterification (2
). Although the functions of Yju2 and HP-X are required only after the action of Prp2, they can be recruited to the spliceosome before the Prp2 step (22
). The findings that the binding of Cwc25 to the spliceosome depends on the function of Prp2 and Yju2 and that the catalytic reaction occurs upon its binding suggest that Cwc25 may play a critical role in the positioning of the 5′ splice site to the branch site. Prp2 requires a cofactor, Spp2, for its binding to the spliceosome and, consequently, for its function (32
), but how Spp2 regulates the function of Prp2 is not known.
In contrast, Cwc22 and Prp2 bind to the spliceosome independently of each other. Neither Cwc22 nor Prp2/Spp2 is associated with the spliceosome in the absence of the NTC, suggesting that these proteins bind only after the spliceosome is activated. Prp2 or Spp2 was not detected to interact with any of the known NTC components by two-hybrid assays (data not shown). Cwc22 is not tightly associated with the NTC but shows weak interactions with Syf3/Ntc77 and Isy1/Ntc30 in two-hybrid assays (data not shown). Whether such interactions are involved in the recruitment of Cwc22 to the spliceosome remains unknown. The stable association of Cwc22 with the spliceosome may involve interactions of Cwc22 with multiple spliceosomal components at a specific stage when the proper conformation of the spliceosome is achieved. Prp2 has been reported to interact with Brr2, which was shown previously to interact with splicing factors involved in various steps of the spliceosome pathway by two-hybrid assays (43
). The interaction of Brr2 with Ntr2 is responsible for the recruitment of the NTR (for NT
elated) complex to the spliceosome to mediate its disassembly (41
). Whether the recruitment of Prp2 is mediated by its interaction with Brr2 also remains to be investigated.
The productive action of Prp2 requires the presence of Cwc22. In the absence of Cwc22, Prp2 could bind to the spliceosome but is dissociated upon ATP hydrolysis, with SF3a/b still retained on the spliceosome. Only in the presence of Cwc22 could SF3a/b be released. Cwc22 thus prevents the spliceosome from entering a futile pathway. How Cwc22 acts in concert with Prp2 to promote the release of SF3a/b remains an open question. It is possible that the binding of Cwc22 may induce a conformational change in the spliceosome to allow the access of Prp2 to SF3a/b or that Cwc22 may interact with Prp2 directly or indirectly to reposition Prp2 from its docking site. Studies of Prp22-mediated mRNA release have revealed a mechanism that involves the repositioning of Prp22 (33
). Prp22 has two roles in the splicing reaction, an ATP-independent role for exon ligation and an ATP-dependent role for mRNA release. Site-specific cross-linking studies have revealed an initial docking of Prp22 on the intron immediately upstream of the 3′ splice site (25
). Prp22 was found to cross-link to mRNA downstream of the splice junction after exon ligation, suggesting that the repositioning of Prp22 during a conformational change of the spliceosome accompanies the second transesterification reaction. Upon ATP hydrolysis, Prp22 moves in the 3′-to-5′ direction to disrupt U5-mRNA interactions in releasing mRNA (33
). Like Prp22, Prp2 exhibits RNA-dependent NTPase activity (18
); however, Prp2 has never been demonstrated in vitro
to unwind RNA duplexes or to disrupt RNA-protein interactions. SF3b is known to bind to the branch site, and components of SF3b cross-link to the branch site (14
). Prp2 has also been shown to directly interact with pre-mRNA by UV cross-linking, likely downstream of the branch site (39
). Whether Prp2 acts by a similar mechanism to disrupt interactions of SF3a/b with the branch site awaits further study.
Unlike Prp2 and Spp2, Cwc22 remains associated with the spliceosome after SF3a and SF3b are released. It is worth noting that Cwc22 is the only factor known that is required for catalytic steps but remains associated after its action. This raises the question of whether Cwc22 has additional roles in subsequent steps of the spliceosome pathway. In the view that Cwc22 modulates the function of Prp2 in the first catalytic step, it will be interesting to know whether Cwc22 also regulates the function of other DExD/H-box ATPases, Prp16 and Prp22, in the second step and for mRNA release, respectively.
Cwc22 has an MIF4G domain at the amino terminus and a MA-3 domain in the middle region. The deletion of the MIF4G domain did not affect cellular growth, whereas the deletion of the middle region containing the MA-3 domain resulted in lethality. The segment of Cwc22 at residues 212 to 453, containing the entire MA-3 domain with extra 80- and 55-amino-acid residues flanking each side, hardly supported cellular growth. The extension of the carboxy-terminal flanking region (to amino acid residue 491) featuring a stretch of the evolutionarily conserved sequence recovered the growth phenotype. Thus, the MA-3 domain together with its flanking regions may constitute a structural domain for the function of Cwc22. Nevertheless, residues 453 to 491 become dispensable in the presence of the MIF4G domain, suggesting a functional redundancy of these two regions.
The MIF4G and MA-3 domains are present in the eukaryotic translational initiation factor eIF4G, in the middle and C-terminal regions, respectively. Each of these domains can interact with DExD/H-box RNA helicase eIF4A in forming the initiation complex (20
). It is tempting to think that Cwc22 may interact with Prp2 in a similar way. Two-hybrid analysis revealed Prp2 to weakly interact with the carboxy-terminal segment of Cwc22 (residues 212 to 577) but not the full-length protein (data not shown). This suggests that although the MIF4G domain is dispensable, its presence affects the interaction of Prp2 with Cwc22 and may also affect their interactions with other spliceosomal components. How such an interaction might affect the function of Prp2 remains to be investigated.