years ago, S. cerevisiae
is believed to have undergone a whole genome duplication (51
). This ancient duplication and subsequent genetic events (e.g., deletions and single-gene amplifications) left the modern yeast with approximately 800 pairs of duplicated genes. In many instances, members of individual gene pairs have evolved distinct yet related biological functions. With this in mind, we used the amino acid sequence of the Prp39p protein to search for and identify a novel component of the yeast U1 snRNP, Prp42p. Prp42p shares 50% amino acid sequence similarity with Prp39p and, like Prp39p, contains multiple copies of the TPR protein recognition domain. The results of this study show that the PRP39
genes are not functionally redundant; each protein coded for by these genes is required at an early stage of splicing to assemble a splicing-competent U1 snRNP. The identification of structurally related, snRNP-specific proteins is unprecedented and indicates that the U1 snRNP is more complex than originally envisioned.
Several lines of evidence implicate Prp42p as a legitimate component of the U1 snRNP. First, the structure of Prp42p is quite similar to that of Prp39p, a protein shown genetically and biochemically to be part of the U1 snRNP complex (26
). Second, an antibody directed against an epitope-tagged version of Prp42p specifically coprecipitates U1 snRNA and supershifts the U1 snRNP in native polyacrylamide gels. Third, the U1 snRNP in extracts depleted of Prp42HAp shows increased electrophoretic mobility and a decreased sedimentation rate, indicative of a change in U1 snRNP structure or stability. Finally, certain mutations within Prp42p are synthetically lethal with a biologically active U1 snRNA deletion derivative (30a
), again consistent with an intimate association between Prp42p and the U1 snRNP.
The absence of Prp42p from a biochemically purified preparation of the yeast U1 snRNP (28
) can possibly be resolved by the observation that the Prp42HAp-U1 snRNP interaction is quite salt sensitive. If the native protein behaves like Prp42HAp, then the 300 mM salt washes used during affinity purification would strip the Prp42p from the U1 snRNP. This apparently weak interaction might reflect a mainly protein-based contact between Prp42p and the U1 snRNP. The snRNP association of yeast U1-C with the U1 snRNP is likely protein mediated and is nearly as salt sensitive as Prp42p (47
). In contrast, the U1-snRNA-binding proteins Snp1p and Mud1p remain bound to the snRNP at NaCl concentrations of at least 200 mM (Mud1p [25
]) and 500 mM (Snp1p [21
]). Whatever the mode of interaction, all available data are consistent with an essential, although possibly salt-sensitive, interaction of Prp42p with the U1 snRNP.
It seems likely that Prp39p and Prp42p are present in the same particle and do not substitute for one another in alternative forms of the U1 snRNP with distinct properties (e.g., distinct substrate preferences). The transcripts of every interrupted gene assayed (i.e., ACT, CYH2, RP51A, and SNR17) required both Prp39p and Prp42p to be spliced in vivo. In addition, the gel shifts associated with anti-HA antibody addition and with Prp42HAp depletion included all of the fully assembled U1 snRNPs independent of whether Prp39HAp or Prp42HAp was being manipulated. If Prp39HAp and Prp42HAp were present in different U1 snRNP populations, these experiments should have revealed a pre-mRNA or snRNP subset insensitive to the manipulation of one or the other factor. Thus, although we have not directly assayed for Prp39p in Prp42p-bearing complexes, no precedent for functionally distinct U1 snRNP subpopulations exists in yeast, and the available data all support Prp39p-Prp42p colocalization in the U1 snRNP.
A consensus element built from the TPR sequences observed in Prp39p and Prp42p is most closely related to that observed in the Drosophila
crn protein (Fig. ). The level of sequence match to the TPR consensus element for the individual Prp39p and Prp42p repeats is low but not unlike that observed for many other TPR elements (10
). In all cases, the Prp39p and Prp42p TPR elements are predicted to present amphipathic alpha-helical surfaces characteristic of the TPR. TPR proteins can be grouped into distinct subfamilies based on their distinctive primary structure features (10
). For instance, domain A of the Drosophila
crn repeats generally has charged residues at positions 6 and 9, aromatic residues at positions 7 and 10, and an aspartic acid residue at position 11. Other than a reduced prevalence of the position 11 aspartic acid, each of these amino acids is well conserved within the Prp39p and Prp42p repeats. Other TPR proteins, including the one other known TPR protein of the spliceosome, Prp6p (22
), show different distributions of amino acids in these positions (10
). The remaining positions from amino acid 1 through the end of domain A of the Prp39p-Prp42p repeats are mostly perfect matches or conservative substitutions for the crn consensus sequence. The domain B structure is less conserved in primary sequence, but individual repeats generally match the crn or an elaborated TPR consensus (52
) at multiple positions and show the overall alpha-helical character of the TPR. In 7 of 11 cases, the predicted domain B alpha helix terminates with a characteristic proline residue (10
) located with a more relaxed positioning between TPR residues 30 and 34.
A number of polypeptide targets for TPR interaction have been identified. For instance, the TPR region of the yeast Cyc8p protein binds to the homeodomain of the yeast α2 protein (43
), while the TPRs of yeast Cdc23p bind to a helix-loop-helix region of Sin1p (38
). In addition, a mutation in the TPR region of the Cdc27p protein reduces its ability to associate with the Cdc23p TPR protein, suggesting a possible TPR-TPR interaction (20
). While these target polypeptides differ considerably in primary sequence, each presents a helical surface for interaction with the corresponding TPR helices. The unique sequence characteristics of the Prp39p and Prp42p repeats (as well as those of other TPR proteins) likely reflect distinctions in the complementary surfaces of their interacting ligands. Genetic screens and direct protein assays are currently under way to identify the natural ligands of Prp39p and Prp42p interaction.
Database comparisons revealed multiple TPRs of the Prp39p-Prp42p-crn domain A sort in only one other protein set, the yeast Rna14p, Drosophila
Su(f), and human CstF77 proteins, in which six to eight repeats were present (this study and reference 50a
). These proteins are part of the RNA cleavage stimulation factor required for pre-mRNA 3′ end processing (see reference 46
and references within). Thus, with the possible exception of crn, all of the known proteins with this variant TPR are involved in RNA processing. Mutations in the Drosophila
crn gene show a pleiotropic embryonic lethal phenotype with impaired neurological (52
) and muscle (7
) development. Based in part on the reduced DNA synthesis observed in mutant embryos, it was suggested that crn may be involved in the cell cycle. We have cloned the likely yeast homolog of crn and are currently investigating its activity in the yeast cell cycle and RNA processing (6a
). A role for yeast crn in splicing or polyadenylation would support the view that the crn-like TPR is reserved for the assembly or intracellular trafficking of complexes involved in RNA maturation.
Prp39p and Prp42p are essential in yeast, while mammalian homologs have not been found. Either these proteins provide a truly yeast-specific function or they are present in mammals but lost in the U1 snRNP isolation procedures used to date. In the first case, the Prp39p and Prp42p proteins might substitute, for instance, for one or more of the mammalian SR proteins absent in yeast but required for early spliceosome assembly events in mammals (e.g., ASF/SF2 and SC35 [reviewed in references 9
]). However, given the general high level of conservation observed between the yeast and mammalian basal spliceosomal components (17
), perhaps a more likely view is that the mammalian equivalents of Prp39p and Prp42p exist but are tenuously associated with the U1 snRNP. The initial failure to identify the phylogenetically conserved, yet weakly bound SF3a and SF3b proteins with the U2 snRNP offers precedent for this (see references in references 17
). The identification of mammalian Prp39p and Prp42p equivalents would reveal the yeast U1 snRNP as more conserved than is suggested by its exaggerated snRNA length.