With the identification of Ntc90p, Ntc77p and Ntc31p, eight components of the Prp19p-associated complex are known. Like previously identified components, these three proteins are associated with the spliceosome in the same way as Prp19p. Thus, all eight known components of the complex bind to the spliceosome at the same time during spliceosome assembly, indicating that the Prp19p-associated complex might be added to the spliceosome as an integral complex. During spliceosome assembly, snRNAs are associated with the spliceosome in a sequential manner and function in splicing in the form of ribonucleoprotein complexes (35
). Although the RNA components of snRNPs play major roles in the recognition and alignment of splice sites through base pair interactions between the snRNAs and intron sequences, some protein components are suggested to perform important functions in modulating structural rearrangement of RNA (14
). Aside from these, no protein splicing factors have been shown to function as a complex. The significance of functional association of the Prp19p-associated complex with the spliceosome awaits further study.
Among the identified Prp19p-associated components, the gene encoding Ntc31p, Ntc30p, Ntc25p or Ntc20p is not essential for cell viability. Accordingly, none of these proteins is essential for splicing, despite their association with the spliceosome during the splicing reaction. The function of these proteins in the splicing machinery remains an interesting question. They may play indirect roles in the splicing reaction through regulating the function of other essential components or they may be functionally redundant to each other or to other splicing factors. Indeed, our previous studies showed that Ntc25p interacts strongly with Prp19p and plays a role in stabilizing the Prp19p-associated complex by modulating the interaction of Prp19p with the other components of the complex (28
). Yeast cells deleted of NTC25
grow poorly at higher temperatures or in synthetic media, reflecting the requirement for Ntc25p under sub-optimal conditions to stabilize the Prp19p-associated complex. Three lines of evidence suggest that Ntc31p, Ntc30p and Ntc20p might have overlapping functions in regulating the function of Ntc90p. First, Ntc31p, Ntc30p and Ntc20p showed identical patterns on interaction with the other components of the Prp19p-associated complex, including Ntc90p. Secondly, these three proteins together formed a stable complex with Ntc90p, as seen in the extract lacking Ntc25p. Finally, genetic analysis revealed that although individual deletion of the NTC31
genes produced no obvious growth defects, deletion of both NTC30
severely impaired cell growth (25
) and additional deletion of NTC31
resulted in cell death. The growth defect of ΔNTC30/ΔNTC20 could be partially rescued by overexpression of NTC90.
In this context, it is likely that Ntc31p, Ntc30p and Ntc20p play roles in modulating interactions of Ntc90p with other components in a similar way to Ntc25p regulation of Prp19p. These three proteins, being in the same subcomplex with Ntc90p, may share common functions in doing so.
are essential for vegetative yeast growth. Prp19p and Ntc85p have been shown to be required for the in vitro
splicing reaction (20
) and NTC90
have been shown to be essential for splicing in vivo
). It is interesting that two of these essential splicing factors, Prp19p and Ntc90p, are regulated by auxiliary components in the complex. These proteins function normally without other cofactors under optimal condition, but otherwise require their regulatory components, possibly to ensure their proper function in accommodating environmental change. This may reflect important functions associated with these factors and may also explain why many genes encoding splicing factors are not essential for viability.
Biochemical analysis reveals that Ntc85p may play a role in promoting binding of the Prp19p-associated complex to the spliceosome (24
). The functional roles of Prp19p, Ntc90p and Ntc77p remain to be investigated. Chung et al.
reported that Clf1p/Ntc77p is required for binding of the tri-snRNP particle to the spliceosome since isolated spliceosomes formed in an in vivo
Clf1p/Ntc77p-depleted extract did not contain U4, U5 or U6 (42
). This result is inconsistent with our finding that Ntc77p, as a component of the Prp19p-associated complex, became associated with the spliceosome after or concurrently with dissociation of U4. In this case, Ntc77p is not required for tri-snRNP binding. The discrepancy between the results of Chung et al
) and ours remains to be resolved.
In an extensive two-hybrid screening, some interactions between components of the Prp19p-associated complex have been identified (30
). The reported results are in part consistent with our identified interactions between components of the complex. This type of analysis, although allowing preliminary establishment of protein linkage networks, does not provide complete information on interactions between designated components which are functionally linked. Indeed, interactions between Ntc85p/Cef1p and Prp19p and between Ntc90p/Syf1p and Ntc77p/Syf3p were not identified in their screening. Instead, our systematic two-hybrid analysis between components of the Prp19p-associated complex has revealed a complete interaction map among all the components. This, in conjunction with the immunoprecipitation analysis, enables us to outline the architecture of the Prp19p-associated complex.
In addition to those interactions we observed among the Prp19p-associated components, interactions between Prp22p and Syf1p/Ntc90p and Syf3p/Ntc77p were also identified by Ben-Yehuda et al
). Prp22p is a member of the DExH protein family and has been demonstrated to be involved in the second step of the catalytic reaction, as well as release of the intron from the spliceosome after completion of the splicing reaction (43
). It was demonstrated that in vitro
depletion of Prp22p resulted in splicing arrest after the first step of the catalytic reaction (44
). In this view, Prp22p is unlikely to be a component of the Prp19p-associated complex. Indeed, western blotting of the purified Prp19p-associated complex using an anti-Prp22p antibody did not detect Prp22p in the complex, although the antibody detected the protein easily in the splicing extract (data not shown). Thus, Prp22p is not tightly associated with the Prp19p-associated complex and interactions between Prp22p and Ntc90p/Syf1p and between Prp22p and Ntc77p/Syf3p might occur only after binding of these components to the spliceosome.