Several fission yeast splicing factors have been identified genetically including Prp1p/Zer1p, Prp2p, Prp4p, Prp5p, Prp8p/Cdc28p, Prp10p, Prp11p, Cdc5p, Dsk1p, Spp13p and Spp42p (2
; T.Tani, personal communication). A few factors were identified by their interactions with the splicing factor Prp2p/U2AF59
, including U2AF25
, Uap2p and Sap155p (Prp10p), using a yeast two-hybrid assay (11
). Srp1p and Srp2p were identified in a search for factors that have arginine/serine-rich regions, a motif found in several mammalian splicing factors (14
). In order to determine the extent of similarity between the fission yeast splicing factors and those of budding yeast and humans, we compared the amino acid sequences using the BLAST 2 Sequences program. The results of this analysis are shown in Table .
Prp1p/Zer1p is more similar to the human protein than the budding yeast protein based on the percent similarity and identity over an extensive stretch of amino acids (Table ). In S.cerevisiae
the homolog of Prp1p/Zer1p is called Prp6p. Prp6p is associated with the tri-snRNP U4/U6.U5. The protein contains 19 tetratricopeptide repeats. Interestingly, the S.pombe prp1-4
strains express a polymorphic phenotype indicating that the gene product is involved in pre-mRNA splicing, poly(A)+
RNA transport and cell cycle progression. The prp13
mutant of fission yeast is synthetic lethal with prp1
). The gene that encodes prp13+
has not yet been cloned, but a suppressor, spp13
has recently been cloned (T.Tani, personal communication). The budding yeast homolog of Spp13p is Prp31p, which has been shown to be associated with the U4/U6.U5 tri-snRNP.
Prp1p also interacts genetically with the Prp4p protein kinase and is phosphorylated in vitro
by Prp4p protein kinase (2
; N.F.Käufer, unpublished data). The Prp4p protein kinase shows 62% similarity over 46% of its amino acids with a human kinase, whereas no S.cerevisiae
kinase with similarity was identified (14
) (Table ). The region of greatest similarity between the fission yeast and human protein comprises a kinase domain plus approximately 150 amino acids at the N-terminal domain. The additional amino acids in the human protein extend the N-terminal domain. Prp4p is essential for growth, and kinase activity is required for pre-mRNA splicing (4
; N.F. Käufer, unpublished data). This study was the first demonstration that a kinase is involved in pre-mRNA splicing.
The Prp4p protein kinase interacts genetically with Spp42p (10
; unpublished data). Spp42p is the homolog of Prp8p (Dbf3p, Slt21p) in budding yeast and p220 in mammals. This protein has been shown to be a bona fide splicing factor in budding yeast and mammals (16
). Spp42p has been highly conserved throughout evolution and Spp42p is slightly more similar to p220 than Prp8p (Table ). Prp8p is thought to be a major player in the switch from inactive to active spliceosome (18
). Surprisingly, Neubauer et al.
did not identify p220 as a spliceosomal protein by mass spectrometry (1
The temperature sensitive (ts) prp5-1 allele is synthetically lethal with the ts prp4-73 allele, suggesting a functional interaction (N.F.Käufer, unpublished data). Prp5p has been evolutionarily conserved between all three organisms we examined, with the fission yeast and human proteins showing the greatest amount of similarity (Table ). The protein contains seven WD domains. Little is known about the function of Prp5p in splicing, but in S.pombe the prp5-1 allele accumulates pre-mRNA and arrests with a 2C DNA content at the restrictive temperature, suggesting that entry into or completion of mitosis is blocked.
encodes an myb-related protein and is essential for G2
/M progression in fission yeast (8
). Because there is good evidence to show that Cdc5p homologs are splicing factors, the phenotype of cdc5-120
was re-examined and the mutant accumulated pre-mRNA at the non-permissive temperature (8
). Both Cdc5p and the S.cerevisiae
homolog, Cef1p, are part of a large complex that has been shown in budding yeast to join the spliceosome when U4 dissociates. It is noteworthy that cdc5-120
is synthetically lethal with prp5-1
and with prp4-73
; N.F.Käufer, unpublished data). Table shows that between the three organisms the protein is evolutionarily conserved with the fission yeast and human proteins showing a greater similarity over a larger region of the protein.
encodes a member of the DExD/H family of putative helicases. The human homolog is more similar over a larger portion of the protein than the budding yeast protein (Table ). In S.pombe
this gene was identified independently as a splicing and cell cycle mutant (6
). The cdc28
mutant produced elongated cells blocked in G2
. The role(s) of Prp8p/Cdc28p in splicing and the cell cycle has not yet been determined.
As discussed above, several of the prp mutants in S.pombe arrest in specific stages of the cell cycle, indicating a link between pre-mRNA splicing and cell cycle progression. This phenotype is not easily explained. In the case of a general splicing factor, one would expect that a stringent mutant phenotype would cause the cells to arrest in all stages of the cell cycle. This is based on the fact that ~45% of the genes in S.pombe, including major cell cycle regulators such as cdc2+, contain introns. A specific cell cycle arrest might indicate that: (i) the protein has two independent functions in cell cycle and pre-mRNA splicing or (ii) the cell cycle block is a consequence of the splicing defect of a transcript needed for cell cycle transitions. In the latter case the protein may be regulating the splicing efficiency of specific pre-mRNAs. So far no study has been able to distinguish between these possibilities to explain the splicing and cell cycle phenotypes.
Prp2p was identified in one of the original screens for splicing mutants (19
). Prp2p encodes the counterpart of the large subunit of U2 auxiliary factor (U2AF65
) in mammals (3
). Using Prp2p/U2AF59
as bait in a two-hybrid assay, a protein was cloned and named U2AF25
because of its similarity to the small subunit of U2AF, referred to as U2AF35
). The large subunits of U2AF are 45% similar and the small subunits are 75% similar over the entire proteins between humans and fission yeast (Table ). Interestingly, the S.cerevisiae
genome apparently does not contain a sequence significantly similar to U2AF59
(based on a Pileup sequence analysis) or U2AF25
, though Mud2p appears to be a functional ortholog of the large subunit of U2AF (20
) (Table ). The U2AF complex is involved in 3′ splice site recognition. U2AF65
binds through its RNA binding domains (RBDs) to the polypyrimidine tract facilitating the interaction of the U2 snRNP with the branchpoint sequence (21
). In three recent studies, it was shown that the 3′ splice site recognition by the U2AF complex is dependent on the architecture of the 3′ end of the intron. For example: introns with very weak polypyrimidine tracts upstream of the AG at the 3′ splice site, require U2AF35
to be spliced properly and the protein interacts directly with the AG (22
The intron architecture, particularly at the 3′ splice site is very different between the two yeasts. Schizosaccharomyces pombe
introns have a degenerate branchpoint consensus sequence CURAY (where R represents purine and Y represents pyrimidine) similar to that present in mammals. In S.cerevisiae
the branchpoint sequence UACUAAC is highly conserved setting it apart from humans and fission yeast. Most fission yeast introns between the branch sequence and the 3′ splice site are pyrimidine-rich and have an average length of 7 nt (25
). However some fission yeast introns are purine-rich while others exhibit a balanced R/Y content in this region, similar to the variability seen in mammalian introns (25
). It is conceivable, therefore, that 3′ splice site recognition in S.pombe
reflects the ancestral splicing machinery, whereas in S.cerevisiae
this part of the machinery has diverged. This notion is consistent with the fact that the small T-antigen intron of the SV40 early region is spliced properly in fission yeast, but not in budding yeast (26
). This intron has the typical architecture of an S.pombe
intron. Therefore, we suggest that the introns of S.pombe
may reflect the architecture of ancestral introns.
Another splicing factor identified in the two-hybrid screen that used Prp2p/U2AF59
as bait was named Uap2p because it was the second U
rotein identified (12
). Uap2p shows 48% similarity with budding yeast Cus2p (Table ). Cus2p is associated with the U2 snRNA in splicing extracts and is postulated to play a role in the proper folding of U2 into a favorable structure prior to spliceosome assembly (27
). Interestingly, although Uap2p and Cus2p have been evolutionarily conserved, the protein–protein interactions between fission and budding yeast were not. Uap2p was identified initially through its interaction with U2AF59
, whereas Cus2p does not interact with the ortholog Mud2p (12
). Uap2p is most similar to the human protein Tat-SF1, especially in the N-terminal domain that contains two RBDs (12
). Tat-SF1 has an acidic C-terminal domain that is reduced considerably in the yeast proteins (27
). Tat-SF1 was originally identified as a transcription factor, but its interactions with other components of the U2 snRNP indicate it may also function in splicing (27
). It has been suggested that the role of Uap2p and its homologs is to mediate the U2AF-dependent association of the U2 snRNP with the intron (27
Prp10p was identified by functional complementation of the prp10-1
strain that accumulates pre-mRNAs at the restrictive temperature (7
). Prp10p is 77% similar to the human protein SAP155 and 67% similar to the budding yeast homolog (Table ). Both yeast proteins are shorter than the human protein. SAP155 is part of the human U2 snRNP (29
). Prp10p has been shown to interact with Prp2p/U2AF59
genetically and in a two-hybrid assay (7
). Whether or not Prp10p plays a role in the recruitment of the U2 snRNP to the 3′ splice site remains to be determined.
Prp11p appears to be the homolog of the S.cerevisiae helicase Prp5p (T.Tani, personal communication). In S.cerevisiae, Prp5p is needed for the base pairing between the U2 snRNP and the branchpoint sequence. The predicted role of Prp5p is the disruption of a Prp9p/U2 snRNP interaction which allows the RNA to refold into a configuration capable of pairing with the intron during pre-spliceosome assembly.
Srp1p and Srp2p are two proteins belonging to the family of SR splicing factors that have been identified and characterized in S.pombe
). These are the first family members of SR splicing factors found in a unicellular organism, therefore a brief discussion is appropriate. Mammals have nine SR proteins consisting of one or two RBDs at the N-terminus and RS/SR dipeptides of different lengths, referred to as the RS domain, at the C-terminus. SR-splicing factors are involved in constitutive and alternative splicing in mammals (30
). All nine SR proteins have at least one RBD containing the submotif RNP-1 that has the highly conserved signature sequence RDAE/DDA. The four SR proteins consisting of two RBDs contain in RBD2 the invariant sequence SWQDLKD. Srp1p and Srp2p contain one and two RBDs, respectively, with the signature sequences. Srp1p has a typical RS/SR domain, whereas Srp2p contains an arginine-rich region including two short SR elements. Overexpression of mutations in the RS/SR domain and the signature sequence RDAE/DDA leads to the accumulation of pre-mRNA indicating an involvement in pre-mRNA processing (14
). Recently the Dsk1p kinase of fission yeast was shown to phosphorylate the RS domains of Srp1p and Srp2p in vitro
). Dsk1p is a homolog of SRPK1. SRPK1 phosphorylates SR proteins like its fission yeast homolog. Comparing Srp1p with the databank revealed that it is closely related to SC35. The two proteins share 29% identity and 41% similarity (Table ). Srp2p is closely related to mammalian SRP40, SRP55 and SRP75 (Table ).
In addition to these two SR family members we also found a gene rsd1
in the S.pombe
databank, encoding a protein which contains an extensive RS domain at the N-terminus followed by three RBDs. This protein has a mammalian counterpart, but no similar protein was found in S.cerevisiae
(Table ). Interestingly, Rsd1p shows the same domain arrangement as Prp2p/U2AF59
and both proteins are phosphorylated in vitro
by Dsk1p (31
). It is presently not known whether Rsd1p plays a role in splicing.