Purification of the yeast U4/U6⋅U5 small nuclear ribonucleoprotein particle and identification of its proteins

Abstract

The yeast U4͞U6⅐U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4͞ U6⅐U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4͞U6⅐U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4͞U6⅐U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.Eukaryotic pre-mRNA must undergo extensive processing in the nucleus before it is converted into the mature mRNA species competent for translation into protein. One important processing step is the excision of intervening sequences, termed introns, which are present within the coding sequences. Removal of introns is effected by the pre-mRNA splicing machinery, which is composed of five phylogenetically conserved small nuclear RNAs (snRNAs) assembled into small ribonucleoprotein particles (snRNPs) and several non-snRNP associated proteins (1). Once assembled onto pre-mRNA, the massive RNP termed the spliceosome removes the intronic RNA sequences by two ordered transesterification reactions ligating the two exons together, forming the mature message. The biochemistry of the mammalian spliceosomal snRNPs has been extensively studied, and many of the protein components have been purified and their genes cloned (2-4). The budding yeast Saccharomyces cerevisiae has been very useful in the study of pre-mRNA splicing, as it is amenable to genetic manipulations. The low abundance of premRNA splicing snRNPs, however, has made their isolation technically challenging. Several yeast proteins involved in splicing have been identified by conditional growth mutations that produce a pre-mRNA splicing defect (5-7) from synthetic lethal analysis (8, 9), identification of second-site suppressors (10-13), two-hybrid analysis (14), and, most recently, from the direct biochemical purification of the yeast U1 snRNP (15,16).In yeast, there are an estimated 100-200 copies of each spliceosomal snRNA (17) as compared with 100,000-1,000,000 snRNA copies in mammalian cells (18). This discrepancy in abundance has made the purification of yeast snRNPs much more difficult, especially in the case of the yeast U4͞U6⅐U5 tri-snRNP, which exists in equilibrium with the individual ...

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

“…Neither protein was reported in the U4/ U6.U5 tri-snRNP particle with Prp38p (Stevens and Abelson 1999;Stevens et al 2001) and we find that fulllength constructs of Cwc23p and Sqs1p fail to interact with Prp38p by the two-hybrid method (data not shown). In addition, when selected under conditions that release Prp38p from the tri-snRNP particle (Xie et al 1998), Prp38p copurifies as a three-component complex with the Spp381p and Snu23p splicing factors without Cwc23p or Sqs1p ( Figure S4).…”

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

“…1+ H-complex (this study)+ 2+ A/B-complex (Bennett et al+, 1992a)+ 3+ A/B-complex (Neubauer et al+, 1998)+ 4+ S. cerevisiae tri-snRNP (Gottschalk et al+, 1999)+ 5+ S. cerevisiae tri-snRNP (Stevens & Abelson, 1999)+ 6+ Human CDC5 complex (Ajuh et al+, 2000)+ 7+ C-complex (this study)+ ⅙ : proteins identified in this study by a single unique peptide only+…”

“…The complexes characterized by Bennett et al+ and Neubauer et al+ both contained a number of proteins involved in early spliceosome assembly, but not found in our C complex preparation+ These include some U1 snRNP proteins and first-step factors (see above)+ B and C complexes do share the core U2 and U5 snRNP proteins, as well as Prp19, SKIP, p68, CDC5, and PRL1+ However, a number of uncharacterized proteins found by Neubauer et al+ were not identified in C complex+ Several proteins shared between B and C complexes were also found associated with the tri-snRNP (U4/U6+U5) from Saccharomyces cerevisiae by mass spectrometry (Gottschalk et al+, 1999;Stevens & Abelson, 1999)+ These studies found the majority of Sm, LSM, and U5 snRNP proteins, as well as several new tri-snRNP proteins+ One of these, SART1, is present in C complex but was not identified in B complex+ The tri-snRNP associated proteins Prp3 and Prp4 are found in B complex, but Prp4 was identified by only a single peptide in C complex+ A U4/U6 associated protein, Prp4 has been shown to join the spliceosome as part of the tri-snRNP, but it appears to depart with U4 snRNA during the transition to C complex (Ayadi et al+, 1997)+ Also listed in Table 2 are proteins associated with the CDC5 protein, first identified as a novel spliceosome component in the mass spectrometry analysis of B complexes (Neubauer et al+, 1998)+ This protein has also been shown to associate with a 40S complex from Schizosaccharomyces pombe where it is essential for pre-mRNA splicing (Burns et al+, 1999;McDonald et al+, 1999)+ The 40S S. pombe complex also contains homologs to U5-220 (Prp8) and U5-116, as well as SmD2, Prp19, Prp5, Syf1, and Ecm2 (Slt11)+ Like C complex, the 40S S. pombe complex contains U2, U5, and U6 snRNAs, but no U1 or U4 (McDonald et al+, 1999)+ Mass spectrometry of immunopurified CDC5 complex from HeLa cells identified Sm proteins and U1 and U2 snRNP proteins, as well as a number of other splicingassociated proteins (Ajuh et al+, 2000)+ In the mammalian system, no U5 or tri-snRNP proteins were identified, although splicing intermediates were immunoprecipitated with CDC5+ Of the proteins associated with mammalian CDC5 that had not been previously linked to splicing, only one, HSP148, was also found in our C complex preparation+…”

The 5??? and 3??? domains of yeast U6 snRNA: Lsm proteins facilitate binding of Prp24 protein to the U6 telestem region

“…To explore the interaction between the U6 telestem and Prp24 protein in yeast, we first investigated whether Prp24 protein is active and important for spliceosome assembly in yeast extracts+ During spliceosome assembly, naked yeast U6 snRNA is complexed by Lsm2-8 and Prp24 proteins to form free U6 snRNP (Stevens et al+, 2001)+ The Prp24 component of free U6 snRNP is thought to be important for promoting duplex forma-59 and 39 domains of yeast U6 snRNAtion between free U6 and free U4 snRNPs to produce U4-U6 di-snRNP and U4-U6•U5 tri-snRNP complexes, at least for Prp24 protein in free U6 snRNPs recycled after a round of splicing (Raghunathan & Guthrie, 1998a)+ During the assembly of U4-U6•U5 tri-snRNP, Prp24 is dissociated from U6 snRNP or is much less tightly associated with it (Shannon & Guthrie, 1991;Gottschalk et al+, 1999;Stevens & Abelson, 1999)+ Formation of U4-U6•U5 tri-snRNP was previously shown to be necessary for spliceosome assembly and splicing (Fetzer et al+, 1997), and tri-snRNP can bind premRNA to assemble spliceosomal complexes in the presence of ATP in vitro (Raghunathan & Guthrie, 1998a)+ However, it was not known whether Prp24 plays a role in the biogenesis of U6 snRNP and tri-snRNP, as studying this process was complicated by the predominance of fully formed and/or recycled U6-containing snRNPs in extract+ In the experiments reported here, endogenous epitope-tagged Prp24(HA) 3 protein in yeast extract was immunodepleted, and this depletion diminished the assembly of 32 P-labeled U6 RNA into de novo tri-snRNPs to 14-16% of mock-depletion levels+ Addition of recombinant Prp24 restored tri-snRNP assembly to 75-97% of mock-depletion levels in replicate assays (Fig+ 6A)+ Endogenous U6 snRNA in these samples was RNase H digested prior to immunodepletion or mock depletion of Prp24 protein, thus to avoid codepletion of U6 snRNP-associated proteins, including Lsm2-8 proteins+ Thus, we found that Prp24 is functional in catalyzing the assembly of naked U6 snRNA into U4-U6•U5 tri-snRNPs during snRNP biogenesis+ Furthermore, this function is important for tri-snRNP assembly in vitro+ Continuing our exploration of the relationship between the U6 telestem and Prp24 protein, we constructed mutant U6 RNAs with mutations that disrupt telestem base pairings+ We also constructed some with additional compensatory mutations that can restore base pairing+ Of those that potentially restore telestem base pairing, there were two types of mutational combinations: one type in which A-U base pairs of the telestem were inverted to U-A pairs, and another type in which A-U base pairs were transverted to G-C pairs that may hyperstabilize the telestem+ The base-pair restoring combinations did show a dramatic restoration of the binding interaction between mutant U6 RNA and Prp24(HA) 3 protein when the compensatory mutations were incorporated in the half of the telestem containing nt 36-39 and 92-95 (see Fig+ 1), that is, when the compensatory mutants U6+36-38polyA,93-95polyU and U6+36-38polyC,93-95polyG were individually assayed for coimmunoprecipitation with Prp24(HA) 3 (Table 4)+ The compensatory effect for these two U6 mutants was 50-fold and 25-fold, respectively+ Parallel controls corroborate that the compensatory effects were dependent on restoration of Watson-Crick base pairing rather than on various combinatio...…”