Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome

Abstract

The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5 (GU) and 3 (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5 GU, and all positions of the 3 YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5 GU. Other mutations in the 5 splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2-to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5 and 3 ends of the intron. This model suggests a mechanism for positioning the 3 splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. The removal of introns from eukaryotic genes to generate mRNA is catalyzed by the spliceosome, a complex RNA-protein machine composed of small nuclear RNAs (snRNAs), and at least 60 proteins. The pre-mRNA splicing reaction consists of two sequential transesterification steps: The first step results in cleavage of the 5Ј splice site (5ЈSS) and formation of a branched (lariat) intermediate; the second step results in ligation of the 5Ј and 3Ј exons. The existence of self-splicing group-II introns, which utilize a similar two-step transesterification mechanism, has led to the hypothesis that premRNA splicing is catalyzed by RNA (Sharp 1985;Cech 1986). Indeed, mutational and cross-linking analyses have revealed a network of RNA-RNA interactions, which have been proposed to form the catalytic core of the spliceosome (Newman 1994;Nilsen 1994).Whether any of the spliceosomal proteins make direct structural or chemical contributions to the active site remains unknown. Numerous cross-linking studies have placed one spliceosomal protein, Prp8, at or near the catalytic core. As summarized in Figure 1, Prp8 has been observed to cross-link to RNA residues within each critical sequence component of the intron: The consensus sequences that define the 5ЈSS, the branch-point (BP), and the 3Ј splice site (3ЈSS) (MacMillan et

Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

“…The multiple recognition events of the 59 splice site make it difficult to explore the contribution from individual factors to the sequence specificity+ To investigate the contribution from other factors than the 59 end of the U1 snRNA, we repeated the selection experiment in the presence of 59 truncated U1 snRNA that could not engage in base pairing with the 59 splice site+ Surprisingly, the consensus 59 splice site sequences after one and three rounds of selection were highly similar to the motifs obtained in the presence of intact U1 snRNA+ It is unlikely that the trace of intact U1 snRNA in the ⌬59end U1-NE contributes significantly to the splicing, as the complex A formed in this extract contained less than 5% intact U1 snRNA+ Moreover, the observed complex A most likely constitutes a functional prespliceosome complex because it was converted into a fully assembled spliceosome (complex B) at the same rate as the appearance of splicing products (Fig+ 7B, right panel)+ Notably, splicing of the 3 ϩ 8 clone in ⌬59end U1-NE reached a level of splicing efficiency after 90 min that was almost indistinguishable from splicing in normal extract+ This suggests that the 59 end of the U1 snRNA is dispensable for splicing and that other factors recognize the entire 59 splice consensus sequence+ Because we, in this study, select for turnover of splicing substrate, the consensus 59 splice site sequence may be shaped both by functional interactions with transacting snRNA and protein factors and as a cisacting sequence with importance for the catalytic step+ One candidate protein factor that potentially could contribute to the sequence specificity is the U1 snRNP specific protein U1C+ Complementation studies with purified U1 snRNP particles lacking subsets of U1-specific proteins show that U1C, but not U1-70K and U1A, is important for formation of early spliceosome complexes in mammalian systems (Heinrichs et al+, 1990;Jamison et al+, 1995;+ Moreover, U1C can be crosslinked to the 59 splice site both in the mammalian (Rossi et al+, 1996) and yeast systems (Zhang & Rosbash, 1999)+ In yeast, the yU1-70K, ySmD1, ySmD3, ySmB, Nam8, and Snu56 proteins also crosslink to the 59 splice site (Zhang & Rosbash, 1999) and the Sm proteins have been shown to stabilize U1 binding (Zhang et al+, 2001)+ Another candidate factor is the U5 snRNP-specific protein Prp8 based on numerous reports of Prp8 crosslinks to the 59 splice site (Wyatt et al+, 1992;Teigelkamp et al+, 1995;Reyes et al+, 1999;Siatecka et al+, 1999;Collins & Guthrie, 1999;Maroney et al+, 2000)+ The binding of Prp8 to the 59 splice site is probably responsible for the recruitment of U4/U6+U5 tri-snRNP to the spliceosome (Konforti & Konarska, 1994) and more recent data suggests that this represents an important ATP-dependent step in early spliceosome assembly (Maroney et al+, 2000)+ During multiple rounds of selection,...…”

Evidence for a role of Sky1p-mediated phosphorylation in 3′ splice site recognition involving both Prp8 and Prp17/Slu4

“…is preferred in wild-type yeast, but the preference is lost in a class of prp8 mutants mapped in the D domain (Umen & Guthrie, 1995b)+ Because sky1⌬ is not synthetically lethal with D domain mutants, it appears unlikely that Sky1p functions in polypyrimidine tract recognition+ This was confirmed by showing that the preference for uridine-rich tract is not impaired in both prp8-39 and sky1⌬ cells (data not shown)+ Prp8 is also known to play a critical role in splicing fidelity as many recessive PRP8 alleles were able to suppress mutations in both 59GT and 39AG dinucleotides (Collins & Guthrie, 1999;Siatecka et al+, 1999)+ Importantly, many of those mutations were mapped to the Prp8 C region, which we have now linked to the allele-specific genetic interaction with sky1⌬ (Fig+ 2)+ It was proposed that the suppression phenotype may result from relaxation of a constraint imposed by Prp8 at the catalytic core (reviewed by Collins & Guthrie, 2000)+ We reasoned that a Sky1p function could also contribute to the normal constraint so that, with a combined effect of prp8-39 and sky1⌬, the loss of this constraint could be lethal+ To test this hypothesis, we determined whether SKY1 contributes to splice site recognition by utilizing an ACT1-CUP1 splicing reporter system, which allows rapid evaluation of cis and trans mutations based on cell growth in the presence of copper sulfate (Lesser & Guthrie, 1993)+ A panel of ACT1-CUP1 reporters containing point mutations at the 59 splice site, the branchpoint, and the 39 splice site were transformed into both wild-type and sky1⌬ cells and the transformants were inoculated onto plates containing increasing concentrations of copper sulfate (see Materials and Methods)+ Indeed, two mutations at the 39AG were modestly but reproducibly suppressed in sky1⌬ cells (Fig+ 4A)+ To confirm the suppression phenotype, we analyzed the reporter RNA by primer extension+ We detected a higher level of the ACT1-CUP1 mRNA from reporter G303C in sky1⌬ than in wild-type cells (Fig+ 4B, compare lanes 7 and 8), but the effect of SKY1 deletion on splicing of reporter A302U was not as obvious (Fig+ 4B, compare lanes 4 and 5)+ The latter is likely due to increased sensitivity of the copper-resistance test compared to primer extension as noted previously (Ben-Yehuda et al+, 2000)+ prp8-39 displayed a similar suppression phenotype by the copper assay (data not shown) as well as by primer extension, especially at position A302 (Fig+ 4B, lanes 6 and 9)+ In contrast, mutations at the 59GT or the branchpoint were not suppressed in sky1⌬ or prp8-39 (Fig+ 4A; data not shown)+ These results indicate that the recognition of 39AG may be the converging point for the concerted action of Prp8 and Sky1p+…”

“…Prp8 is highly conserved between yeast and mammals and is known to form multiple contacts with conserved splicing signals in both pre-mRNA and snRNAs in the spliceosome (reviewed by Collins & Guthrie, 2000)+ However, this largest known splicing factor has no recognizable protein motifs+ To aid in deciphering its function, the protein has been arbitrarily divided into four domains, A to D, for PCR mutagenesis, and a large number of recessive alleles were mapped into clusters within the B, C, and D domains by the Guthrie and Konarska groups (listed in Fig+ 2A; Umen & Guthrie, 1996;Collins & Guthrie, 1999;Siatecka et al+, 1999)+ For example, alleles D101-107 and C121-125 were previously characterized as suppressors of mutations in the polypyrimidine tract and the 39YAG, respectively (Umen & Guthrie, 1995b)+ More recently, many prp8 alleles were found to simultaneously suppress mutations in both the 59GU and 39YAG (Collins & Guthrie, 1999;Siatecka et al+, 1999)+ Characterization of these prp8 alleles has led to functional assignment of Prp8 in spliceosome assembly and splice site recognition (Brown & Beggs, 1992;Collins & Guthrie, 1999;Siatecka et al+, 1999)+ More recently, characterization of a novel prp8 mutant (known as prp8-201, a U4-cs1 suppressor) indicates that Prp8 is also critical for RNA rearrangement (Kuhn et al+, 1999;Kuhn & Brow, 2000)+ We therefore asked whether Sky1p function could be related to a specific pathway involving Prp8 by surveying previously characterized prp8 alleles with regard to their genetic interaction with the kinase+ A large collection of prp8 alleles was surveyed for possible genetic interactions with sky1⌬ (Fig+ 2B)+ Two alleles located in the C domain (C133 and C134) were found to be synthetically lethal with deletion of SKY1+ Interestingly, the newly identified prp8-39 mutation was also mapped in the C domain near the mutations in C133 and C134 (Fig+ 2A)+ None of the B and D domain alleles including the recently described prp8-201 displayed synthetic lethality with sky1⌬ (Fig+ 2B; A+ Kuhn, unpubl+ observation)+ Importantly, the analysis also revealed that a ts phenotype is not a prerequisite for the genetic interaction between PRP8 and SKY1: C134 was synthetically lethal with sky1⌬, but did not display a ts phenotype+ On the other hand, a number of ts alleles in the C and D domains were not synthetically lethal with sky1⌬+ Together, these results establish an allele-specific genetic interaction between SKY1 and PRP8 and suggest that Sky1p may play a role in premRNA splicing in a specific pathway involving a small region in the C domain of Prp8+ SAD1 overexpression suppresses the splicing defect in prp8-39 but not the synthetic lethality with sky1D…”

“…SKY1 functionally interacts with PRP8 in an allele-specific manner+ A: Diagram of the PRP8 gene+ Shown above are prp8 alleles in B, C, and D domains+ Thick lines indicate the regions containing the mutations+ The prp8-39 mutation was mapped in the C domain as shown (arrow)+ B: Summary of the genetic interaction between SKY1 and the PRP8 alleles tested+ The prp8 mutations were previously described (Umen & Guthrie, 1996;Collins & Guthrie, 1999)+ yJU71-sky1⌬ was transformed with prp8 alleles and the transformants were screened for viability on 5-FOA+ The degree of synthetic lethal (SL) with sky1⌬: Ϫ: wild-type growth; ϩϩ: appearance of some small colonies after 1 to 2 weeks of culturing; ϩϩϩ: no growth+ 1288 S.F. Dagher and X.-D. Fu either in the assembly of functional U4/5/6 tri-snRNP before splicing or during RNA rearrangement within the spliceosome+ However, prp8-39 remained synthetically lethal with sky1⌬ regardless of SAD1 overexpression (Fig+ 3C)+ Thus, the ts phenotype associated with prp8-39 may be distinct from its genetic interaction with SKY1+ More importantly, this result in conjunction with the lack of genetic interaction between prp8-201 and sky1⌬ implies that the synergy between Sky1p-mediated phosphorylation and Prp8 may not occur at the level of snRNP assembly and rearrangement during splicing+…”

“…Yeast strains used in the current study are listed in Table 1+ The strain used for the synthetic lethal screen (CH1305) was a gift from the Holm laboratory (Kranz & Holm, 1990)+ SKY1 was deleted from all strains by using a kanamycin-resistance expression unit flanked by SKY1 genomic sequence as previously described (Yeakley et al+, 1999)+ The plasmid pCH1675-SKY1 was constructed by inserting the BamHISal I fragment containing SKY1 from pRS316-SKY1 (Siebel et al+, 1999) into the BamHI-SmaI sites of pCH1675 (Kranz & Holm, 1990)+ Wild-type SKY1 was similarly subcloned into the LEU2-marked plasmid YCpLac111+ The mutant YCpLac111-sky1 K-M was constructed by exchanging the PshAI-Pfl MI fragment from YCpLac111-SKY1 with the corresponding fragment from pYES2-sky1 K-M (Siebel et al+, 1999)+ The prp8 null strains yJU71 and yJU75 used for allele specificity and splice site suppression analyses were gifts from the C+ Guthrie laboratory+ The slu4 [ySJ134 (prp17-1), ySJ136 (prp17⌬ )] and slu7 [yDFA7C (slu7-1)] mutant strains, and plasmids pSJ3 (wild-type SLU4 in YCp50) and pDF64 (wild-type SLU7 in YCp50) were also gifts from the C+ Guthrie laboratory+ The prp18 null strain DH120R and the plasmid pDH101 bearing wild-type PRP18 were gifts from the D+ Horowitz laboratory+ The prp16 (prp23-ts514) and prp22 (prp22-ts107) mutant strains were gifts from the J+ Abelson laboratory (Vijayraghavan et al+, 1989)+ The URA/CEN plasmids carrying PRP16 (pS32) and PRP22 (p360) were gifts from the B+ Schwer laboratory+ All wild-type and mutant PRP8 alleles listed in Figure 2 and all ACT-CUP1 reporters used in Figure 4 were kindly provided by the C+ Guthrie laboratory (Lesser & Guthrie, 1993;Umen & Guthrie, 1996;Collins & Guthrie, 1999)+ Synthetic lethal screen SKY1 was first disrupted in CH1305 and the resulting SLsky1⌬ strain was covered with pCH1675-SKY1 (CEN, ARS, URA3, ADE3 )+ UV mutagenesis conditions were chosen to produce 25-30% viability on YP galactose plates+ Nonsectoring cells were isolated from approximately 100,000 surviving colonies and then subjected to 5-FOA selection+ Three synthetic lethals (SL34, SL39, and SL44 ) were obtained and all showed ts growth at 37 8C+ SL34 and SL44 had high reversion rates on 5-FOA plates following long incubation+ In contrast, SL39 was stable and therefore chosen for further functional studies+ The SL39 gene was cloned by complementing the ts phenotype with the Heiter yeast genomic DNA library, and identified as PRP8 by partial sequencing+ The prp8-39 mutation was determined by gap repair (Guthrie & Fink, 1991)+ Briefly, a series of restriction fragments was prepared from pSE363-PRP8 (HIS3, CEN, ARS ) (a gift from the C+ Guthrie laboratory) and transformed into the SL39 strain+ All but the PshAI-SpeI fragment were able to correct the ts phenotype, indicating the SL39 mutation(s) resides in this region+ The PshAI-SpeI gapped pSE363-PRP8 was then transformed into the SL39 strain and the repaired plasmid was isolated+ Sequencing of the PshAI-SpeI region in the p...…”