In S. pombe
, S. cerevisiae
, and human cells, Cdc5p, Cef1p, and hCDC5, respectively, have been implicated in the process of pre-mRNA splicing and are components of multiprotein complexes (3
). In this study we analyzed S. pombe
Cdc5p- and S. cerevisiae
Cef1p-associated proteins using TAP (55
) coupled with DALPC tandem mass spectrometry. This powerful approach revealed the existence of a stable complex containing at least 26 proteins. The majority of these proteins are known components of the U2 or U5 snRNPs or have otherwise been implicated in the process of pre-mRNA splicing, although several uncharacterized proteins were also identified. This work marks the first comprehensive analysis of CDC5-associated proteins in yeasts and provides evidence that the members of this complex must cooperate in some aspect of pre-mRNA processing.
We have confirmed the protein associations detected by DALPC by conventional methods. In all cases we have tested, at least a portion of each identified protein is able to coimmunoprecipitate with either Cdc5p or Cef1p. In addition, we have tested three of the uncharacterized proteins for a role in pre-mRNA splicing. We have found that the essential component (S. pombe Cwf7p) is required for pre-mRNA splicing, and genetic analysis of the nonessential components (S. pombe Cwf11p and S. cerevisiae Cwc15p) suggests that they positively contribute to Cdc5p/Cef1p function. These results suggest that all components identified in our purifications function in the process of pre-mRNA splicing.
One striking result of the DALPC analysis is the conservation between the S. pombe
Cwf complex and the S. cerevisiae
Cwc complex. These complexes contain similar numbers of components, with 24 proteins being conserved between species. Significantly, homologs of all Cwf proteins previously identified in S. pombe
were found in each S. cerevisiae
purification, and homologs of all S. cerevisiae
Ntc components were identified in the S. pombe
Cdc5p purification, with a few exceptions. The exceptions were those proteins without obvious counterparts in the other yeast (S. pombe
Cwf7p, S. cerevisiae
Snt309p [Ntc25p], and S. cerevisiae
Ntc20p). Homologs of S. cerevisiae
Snt309p and Ntc20p are also not obvious in higher eukaryotes, but there is a mammalian homolog of S. pombe
Cwf7p, termed SPF27, that copurified with hCDC5 (3
The S. cerevisiae
Ntc complex includes Prp19p, Cef1p (Ntc85p), Snt309p (Ntc25p), Isy1p (Ntc30p), Ntc20p (11
), and at least six other unidentified proteins (Ntc120p, Ntc90p, Ntc81p, Ntc77p, Ntc50p, and Ntc40p). A number of proteins identified here could represent these unknown polypeptides. For example, the 40-kDa S. cerevisiae
Cwc2p is probably Ntc40p, S. cerevisiae
Snu114p might represent Ntc120p, S. cerevisiae
Syf1p might represent Ntc90p, S. cerevisiae
Clf1p could correspond to Ntc81p, and S. cerevisiae
Cwc1p/Prp46p may represent Ntc50p.
The identification of Ntc components within the Cdc5p-TAP purification strongly suggests that Ntc components are members of a larger protein complex. DALPC analysis of Prp19p-TAP- and Snt309p-TAP-associated proteins further strengthens this conclusion. Both of these S. cerevisiae
Ntc members copurified with the same contingent of polypeptides as did S. pombe
Cdc5p-TAP, S. cerevisiae
Cef1p-TAP, S. cerevisiae
Cwc1p-TAP, and S. cerevisiae
Cwc2p-TAP. Like S. pombe
Cdc5p-TAP, S. cerevisiae
Prp19-TAP was also found to associate with the U2, U5, and U6 snRNAs. Therefore, Ntc proteins might represent a smaller unit of more stably associated proteins. It should be noted, however, that our DALPC analyses are not quantitative and do not provide information on the stoichiometry of any component within the larger complex we have examined. Therefore, it is possible that a smaller complex(s) does exist as a separate unit in cells, especially in S. cerevisiae
, and joins with other proteins at certain times during the pre-mRNA splicing reaction. However, we have no data to support this interpretation from our studies in S. pombe
. Under all conditions we have tested, Cdc5p exists in a large, discrete complex that contains numerous polypeptides and all three snRNAs (Fig. ; Table )(43
). An alternative explanation for the existence of a smaller unit in S. cerevisiae
is that the larger complex might simply be less stable in the buffer conditions employed for its purification than its S. pombe
counterpart. If so, by either breaking apart during cell lysis, sucrose gradient fractionation, or purification, several S. cerevisiae
complex members could behave as if they were in smaller complexes.
The conservation of Cdc5p/Cef1p complexes between yeasts raises the possibility that CDC5 complexes in higher eukaryotes also contain similar proteins. Human CDC5 has been shown to copurify with a core complex of six proteins, as well as a number of other polypeptides (3
). Four of the mammalian core proteins correspond to proteins identified in the yeast complexes (S. pombe
Cdc5p and S. cerevisiae
Cef1p; S. pombe
Cwf8p and S. cerevisiae
Prp19p; S. pombe
Cwf7p; and S. pombe
Prp5p/Cwf1p and S. cerevisiae
Prp46p/Cwc1p). However, homologs of the majority of hCDC5-copurifying proteins were not found in the yeast preparations. None of the hCDC5-copurifying proteins was tested for its interaction with hCDC5 by direct coimmunoprecipitation or other follow-up methods (3
), and therefore, their reported association with hCDC5 might be misleading. On the other hand, our fairly comprehensive analysis of the yeast Cdc5p/Cef1p complexes represents the minimum complement of Cdc5p/Cef1p-associated proteins. Only proteins whose binding is not altered by the presence of Ca2+
and that remain stably associated through the two affinity purification steps can be identified by the TAP strategy. Thus, it is likely that still other proteins will be found to interact with those identified in this study.
The detection of the U2, U5, and U6 snRNAs along with core Sm proteins, U2 snRNP components, and U5 snRNP components in the Cwf/Cwc complexes has led us to speculate that Cdc5p/Cef1p and associated proteins are a part of, or equivalent to, the active, mature form of the spliceosome. Such a conclusion is consistent with in vitro studies in which hCDC5 was found associated with core Sm proteins throughout the pre-mRNA splicing reaction (10
). It is possible that many of the novel proteins identified in our purifications will be found to be members of the U6 snRNP, which has yet to be well characterized in any organism. Structural analysis of individual snRNPs and core Sm-protein complexes have confirmed that snRNAs found in an assembled spliceosome would be buried in the center of a protein core (17
). The stability of the Cwf complex in the presence of RNase A provides further evidence that that the Cwf/Cwc complex could represent the active spliceosome.
Although this report is the first to link all of the proteins listed in Table into one stable unit, many genetic and physical interactions between S. cerevisiae
Cwc components have been noted previously (6
). Also, a number of S. cerevisiae
Cwc components are known to interact with U2, U5, and/or U6 snRNAs either by photo-cross-linking or by coimmunoprecipitation experiments (18
). Cef1p, Prp19p, Snt309p, Isy1p/Ntc30p, and Ntc20p associate with U2, U5, and U6 snRNPs in vitro, concomitant with U4 snRNP dissociation (11
). Other S. cerevisiae
Cwc components, such as Brr2p, Prp8p, Snu114p, Clf1p, and Slt11p/Ecm2p, are known to initiate and/or stabilize key RNA-RNA and protein-RNA transitions during pre-mRNA splicing (5
). Taken in combination, these data provide further support for the idea that the complex described here represents a portion of the active spliceosome. Indeed, it is possible that the Cdc5-TAP complex simply represents U2, U5, and U6 snRNPs containing proteins not previously recognized as U2, U5 and U6 snRNP components.
It was unexpected, however, that our attempts to prevent formation of the Cdc5p complex by blocking pre-mRNA splicing using a variety of pre-mRNA splicing mutants were not successful. In all cases tested, Cdc5p was detected only in a high-molecular-weight complex, indistinguishable from the complex present in wild-type cells. This could indicate that at steady state, most of the pre-mRNA splicing factors associated with Cdc5p are actively engaged in the splicing reaction and only transiently dissociate to perform the initial pre-mRNA recognition steps. Given the relative stability of the purified S. pombe Cdc5p-TAP complex, further analysis of its RNA composition and structure should help explain these observations and provide insight into its role in pre-mRNA processing.