Pti1p and Ref2p found in association with the mRNA 3' end formation complex direct snoRNA maturation
2003; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.1093/emboj/cdg253
ISSN1460-2075
AutoresSonia Dheur, Le Thuy Anh Vo, Florence Voisinet-Hakil, Michèle Minét, Jean‐Marie Schmitter, François Lacroute, Françoise Wyers, Lionel Minvielle-Sébastia,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 June 2003free access Pti1p and Ref2p found in association with the mRNA 3′ end formation complex direct snoRNA maturation Sonia Dheur Sonia Dheur Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Le Thuy Anh Vo Le Thuy Anh Vo Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Florence Voisinet-Hakil Florence Voisinet-Hakil Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Michèle Minet Michèle Minet Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Jean-Marie Schmitter Jean-Marie Schmitter Institut Européen de Chimie et Biologie, CNRS, FRE 2247, 33607 Pessac, cedex, France Search for more papers by this author François Lacroute François Lacroute Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Françoise Wyers Françoise Wyers Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Lionel Minvielle-Sebastia Corresponding Author Lionel Minvielle-Sebastia Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Sonia Dheur Sonia Dheur Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Le Thuy Anh Vo Le Thuy Anh Vo Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Florence Voisinet-Hakil Florence Voisinet-Hakil Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Michèle Minet Michèle Minet Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Jean-Marie Schmitter Jean-Marie Schmitter Institut Européen de Chimie et Biologie, CNRS, FRE 2247, 33607 Pessac, cedex, France Search for more papers by this author François Lacroute François Lacroute Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Françoise Wyers Françoise Wyers Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Lionel Minvielle-Sebastia Corresponding Author Lionel Minvielle-Sebastia Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Author Information Sonia Dheur1, Le Thuy Anh Vo2, Florence Voisinet-Hakil1, Michèle Minet2, Jean-Marie Schmitter3, François Lacroute2, Françoise Wyers2 and Lionel Minvielle-Sebastia 1 1Institut de Biochimie et Génétique Cellulaires, CNRS, UMR 5095, 1 Rue Camille Saint Saëns, 33077 Bordeaux, cedex, France 2Centre de Génétique Moléculaire, CNRS, UPRA 2167, 91198 Gif-sur-Yvette, cedex, France 3Institut Européen de Chimie et Biologie, CNRS, FRE 2247, 33607 Pessac, cedex, France ‡S.Dheur and L.T.A.Vo contributed equally to this work *Corresponding author. E-mail: [email protected]c.u-bordeaux2.fr The EMBO Journal (2003)22:2831-2840https://doi.org/10.1093/emboj/cdg253 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3′ ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3′ end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3′ end processing complex, are essential for yeast snoRNA 3′ end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3′ end formation of non-polyadenylated transcripts. Introduction mRNA 3′ end formation is achieved in two coupled reactions; cleavage followed by polyadenylation of the precursor (for a recent review see Edmonds, 2002). A large protein complex comprising the cleavage and polyadenylation factors CF IA (cleavage factor IA), CPF (cleavage and polyadenylation factor), Nab4p and Nab2p has been identified which can recapitulate the processing reaction in vitro on synthetic pre-mRNA substrates (Minvielle-Sebastia et al., 1994, 1998; Kessler et al., 1996, 1997; Ohnacker et al., 2000; Hector et al., 2002). CF IA is a tetrameric factor that consists of Rna14p, Rna15p, Pcf11p and Clp1p (Minvielle-Sebastia et al., 1997; and references therein). CPF contains multiple subunits ranging in size from 150 to 20 kDa. The first definition of this factor (Ohnacker et al., 2000) assigned the nine following polypeptides to it: Yhh1p/Cft1p (150 kDa; Stumpf and Domdey, 1996; Preker et al., 1997), Ydh1p/Cft2p (105 kDa; Preker et al., 1997; Zhao et al., 1997), Ysh1p/Brr5p (100 kDa; Chanfreau et al., 1996; Jenny et al., 1996; Preker et al., 1997), Pta1p (90 kDa; Preker et al., 1997), Pap1p (64 kDa; Lingner et al., 1991; Preker et al., 1997), Mpe1p [58 kDa; Vo et al., 2001; note that this polypeptide was referred to originally as Pfs1p in Preker et al. (1997) and Ohnacker et al. (2000)], Pfs2p (53 kDa; Preker et al., 1997; Ohnacker et al., 2000), Fip1p (50 kDa; Preker et al., 1995) and Yth1p (26 kDa; Barabino et al., 1997). Recently, pre-mRNA 3′ end processing could be reconstituted in vitro with CF IA and CPF purified from protein A-tagged subunits, the latter containing six additional polypeptides compared with previous descriptions of the factor. They were identified as Swd2p, Glc7p, Ssu72p, Pti1p, Ref2p and a 20 kDa protein encoded by the YDL094c open reading frame (ORF) (Dichtl et al., 2002). Among them, only Ref2p has been suggested to be directly involved in pre-mRNA 3′ end processing (hence its name, RNA end formation; Russnak et al., 1995). Yeast small nucleolar RNAs (snoRNAs) are also subject to 3′ end cleavage to provide an entry site for exonucleases that produce mature 3′ ends by exonucleolytic trimming (Chanfreau et al., 1998a,b; Allmang et al., 1999; van Hoof et al., 2000; Perumal and Reddy, 2002). In some instances, 3′ entry sites are generated by recognition of a specific stem structure by the RNA endonuclease Rnt1p (Chanfreau et al., 1998a,b). However, many mono- and polycistronic snoRNAs do not bear a Rnt1p-specific signal in their 3′ region. Recently, some cis-acting elements and trans-acting factors have been identified in yeast that participate in 3′ end formation of at least some snRNAs and snoRNAs. The Nrd1 protein, which interacts with the C-terminal domain (CTD) of RNA polymerase II (pol II), the RNA-binding protein Nab3p, the CTD kinase Ctk1p, and Sen1p have all been shown to function in snRNA and snoRNA 3′ end maturation (Conrad et al., 2000; Steinmetz et al., 2001). More surprisingly, mutations in RNA14 and RNA15 genes that code for CF IA subunits (Minvielle-Sebastia et al., 1994) have been described to inhibit poly(A)-independent 3′ end processing of U2 and U5 pre-snRNAs and also of different box C/D and box H/ACA pre-snoRNAs (Fatica et al., 2000; Morlando et al., 2002). However, the CF IA-complementary factor CPF (Barabino et al., 2000; Ohnacker et al., 2000; Dichtl and Keller, 2001) has been proposed not to participate in this processing since none of the CPF mutants tested so far were affected in small RNA 3′ end processing (Dichtl et al., 2002; Morlando et al., 2002). Together, these results suggested that CF IA might work in combination with a set of proteins distinct from CPF, for instance the Nrd1p-associated polypeptides, to achieve poly(A)-independent 3′ end formation of sn(o)RNAs (Steinmetz et al., 2001; Morlando et al., 2002). However, we show here that two newly discovered CPF-associated proteins, Pti1p and Ref2p, are involved in cleavage-dependent/polyadenylation-independent 3′ end formation of snoRNAs, therefore demonstrating that most of the pre-mRNA 3′ end processing machinery participates in independently transcribed snoRNA 3′ end formation. Results PTI1 is a multicopy suppressor of a pcf11 temperature-sensitive mutant The four components of CF IA are encoded by the RNA14, RNA15, PCF11 and CLP1 genes. Mutant alleles of each of these genes are deficient for cleavage and polyadenylation in vitro (Minvielle-Sebastia et al., 1994, 1997; Amrani et al., 1997; our unpublished data). To extend our knowledge of the reaction, we sought multicopy suppressors of the pcf11-2 temperature-sensitive mutant. We transformed pcf11-2 cells with a multicopy plasmid-borne genomic library and looked for temperature-resistant clones at 37°C. About 30% of the thermoresistant clones tested carried inserts sharing the putative ORF YGR156w called PTI1 in the Saccharomyces cerevisiae genome database (SGD; http://genome-www.stanford.edu/Saccharomyces/). After subcloning PTI1 individually into a new multicopy vector (pFL44; Bonneaud et al., 1991), we could still observe suppression of the pcf11-2 temperature-sensitive phenotype (Figure 1A). Therefore, overexpression of PTI1 was sufficient to allow growth at 37°C of the otherwise temperature-sensitive pcf11-2 allele. PTI1 is an essential gene that codes for a predicted protein of 425 amino acids, well conserved in eukaryotes. Peculiar features were identified in Pti1p such as an N-terminal RRM-type RNA-binding domain and several potential phosphorylation sites. Remarkably, database searches assigned the 64 kDa subunit of cleavage stimulation factor (CstF) as a putative mammalian homologue of Pti1p (see SGD). Also, Pti1p showed significant similarities with the CF IA subunit Rna15p, which has been found itself to be very similar to CstF-64K (Takagaki and Manley, 1994). Figure 1.Phenotypic analysis of pcf11-2, pti1-2, ref2-2 and ref2Δ mutants. (A) Temperature sensitivity of the pcf11-2 mutant can be suppressed by overexpression of PTI1 or REF2 cloned on a multicopy plasmid (pPTI1 and pREF2, respectively). In addition, PTI1 and REF2 are multicopy suppressors of ref2-2 and pti1-2, respectively. (B) REF2 is essential for viability in the W303 yeast strain background. Tetrads from a REF2/ref2Δ::TRP1 heterozygote were dissected on rich medium and incubated at 22°C for at least a week. Two spores only were viable under these conditions, even after a prolonged incubation time (3 weeks). (C) Sporulation of a diploid strain heterozygous for the REF2::TRP1 disruption (lane 1, 2n) gave two spores bearing the deletion which were viable on medium lacking tryptophan provided they were transformed with a plasmid-borne wild-type REF2 gene (lanes 2 and 3, spores A and B, middle row). Those spores were unable to grow on a 5-fluoro-orotic acid (5-FOA)-containing medium since the URA3-marked complementing plasmid was lost when cells were cultivated in the presence of this drug (lanes 2 and 3, upper row). In contrast, the two non-disrupted spores were inviable on medium lacking tryptophan but were not affected by 5-FOA (lanes 4 and 5, spores C and D, middle and upper rows, respectively). Download figure Download PowerPoint We next screened two-hybrid libraries with PTI1 as bait. This technique allows for the detection of protein–protein interactions in vivo (Fields and Song, 1989). Among the preys obtained, the most significant clones corresponded to PTA1 and REF2 genes (10 independent clones for both genes with seven different inserts each time). Pta1p has already been identified as an intrinsic subunit of CPF (Preker et al., 1997), whereas Ref2p has been described originally as an enhancer of usage of weak poly(A) sites (Russnak et al., 1995). Consistently, multicopy suppression of pcf11-2 temperature sensitivity was also obtained with REF2 at 37°C (Figure 1A). These genetic data suggested that PTI1, like REF2 and PTA1, could code for proteins involved in mRNA 3′ end formation. We subsequently investigated whether PTI1 and REF2 were essential for cell viability in the wild-type strain W303. We confirmed that disruption of the PTI1 ORF led to lethality (data not shown; see also SGD). Unexpectedly, we also found REF2 to be essential for vegetative growth at all temperatures on rich medium, which is the opposite finding to previous analyses of the gene (Russnak et al., 1995; Winzeler et al., 1999). Indeed, diploid heterozygotes REF2/ref2Δ::TRP1 were sporulated, and tetrad analysis gave rise to two viable, tryptophan-auxotroph spores (therefore carrying the non-disrupted wild-type REF2 gene) and two lethal spores (Figure 1B). The non-viable spores corresponded to the ref2::TRP1 disruptants since they can survive on medium lacking trytophan provided they are rescued by the plasmid-borne REF2 gene (Figure 1C, spores A and B; see also Materials and methods). This discrepancy could be explained by the different genetic background used in our study. In addition, it should be recalled here that only one-third of the REF2 gene was replaced by the LEU2 marker in Russnak et al. (1995). This deletion resulted in a slow growth phenotype that could therefore be attributed to the production of a truncated, albeit still partially functional protein. These results demonstrated that REF2 as well as PTI1 were essential for viability of W303. Pti1p and Ref2p are not essential for pre-mRNA 3′ end processing in vitro It has been shown that extracts prepared from pre-mRNA 3′ end processing mutants generally exhibit cleavage and/or polyadenylation defects as tested in vitro (e.g. Minvielle-Sebastia et al., 1994; Preker et al., 1997). We thus generated conditional lethal mutants by subjecting PTI1 and REF2 to mutagenic PCR to gain more insight into their potential role in mRNA 3′ end formation. The temperature-sensitive alleles pti1-2 and ref2-2 obtained were unable to grow at 37°C but survived at 24°C, although exhibiting a slow growth phenotype (Figure 1A). Interestingly, overexpression of PTI1 and REF2 suppressed cell lethality of ref2-2 and pti1-2 at 37°C, respectively, suggesting again that they have related functions (Figure 1A). We then prepared extracts competent for pre-mRNA 3′ end processing in vitro from wild-type and mutant pti1-2 and ref2-2 strains as described (Minvielle-Sebastia et al., 1994). Unexpectedly, we found pti1-2 and ref2-2 mutant extracts to be as active in cleavage and polyadenylation as the wild-type extract (data not shown). The putative role of Pti1p and Ref2p in mRNA maturation was investigated further by seeking their association with the 3′ end processing complex. We performed tandem affinity purification (TAP method; Rigaut et al., 1999) of CPF from PFS2-TAP- and MPE1-TAP-expressing strains as both Pfs2p and Mpe1p have been identified as CPF subunits (Ohnacker et al., 2000; Vo et al., 2001). Mass spectrometry analysis of the factors revealed that both Pti1p and Ref2p were associated with CPF (our data, not shown), as has been suggested recently in a large-scale analysis of yeast protein complexes (Gavin et al., 2002). We subsequently purified CPF from the wild-type and pti1-2 and ref2-2 mutant strains in which the Fip1p subunit was N-terminally TAP tagged (see Materials and methods). The factors obtained showed band patterns on protein gels similar to each other and to those previously described following purification by alternative methods (Figure 2A; Preker et al., 1997; Ohnacker et al., 2000; Vo et al., 2001; Dichtl et al., 2002). However, Pti1-2p and Ref2-2p were not detectable by western blotting in the mutant factors whereas they were present in the processing extracts (see Supplementary data available at The EMBO Journal Online). They nevertheless were assayed in vitro for pre-mRNA 3′ end cleavage in combination with wild-type CF IA and recombinant Nab4p (Figure 2B) and for polyadenylation in association with CF IA, Nab4p and Nab2p (Figure 2C; Minvielle-Sebastia et al., 1998; Hector et al., 2002). As seen with extracts, efficient cleavage of the CYC1 pre-mRNA (Figure 2B, lanes 2–4) and polyadenylation of the pre-cleaved substrate CYC1pre (Figure 2C, lanes 4–6) could be observed with either the wild-type or pti1-2 and ref2-2 purified factors. These activities were retained even at higher temperature (34°C; data not shown), strongly supporting that Pti1p and Ref2p were not essential for pre-mRNA 3′ end processing in vitro. Figure 2.Reconstitution of pre-mRNA 3′ end processing activity with purified factors. (A) Silver-stained gel of purified CPF obtained from TAP-tagged Fip1p in the wild-type (WT) or pti1-2 and ref2-2 mutant background. M, molecular weight marker, in kDa. (B) Cleavage assays with purified CF IA, recombinant Nab4p and the different TAP factors purified as described in Materials and methods. Lanes 1 and 5, unreacted iso-1-cytochrome c precursor (CYC1); 5′, 3′, upstream and downstream cleavage products, respectively. Cryptic cleavage products are indicated by asterisks (Minvielle-Sebastia et al., 1998). (C) Polyadenylation of the CYC1 pre-cleaved RNA (CYC1pre) which ends at the natural polyadenylation site. Assays were performed with purified CF IA, recombinant Nab2p, Nab4p and Pap1p (where indicated), and the different TAP factors purified as described in Materials and methods. Lane 1, unreacted CYC1pre precursor; lane 3, 10 ng of Pap1p were added; lane 8, 20 ng of Pap1p were added. pA, polyadenylated products. M, labelled MspI-digested pBR322 serves as molecular weight markers, in number of nucleotides. (D) Western blot analysis of the different TAP factors. The membranes were probed with polyclonal antibodies to either Pap1p (at a 1:2000 dilution) or Fip1p (diluted at 1:10 000). CBP-Fip1p refers to as the calmodulin-binding peptide-tagged Fip1 protein obtained after TEV protease cleavage during TAP purification (Rigaut et al., 1999). Download figure Download PowerPoint To test more directly whether Pti1p- and Ref2p-associated components are competent for mRNA 3′ end formation, we purified the Pti1p-TAP and TAP-Ref2p factors (see Materials and methods). The isolated complexes were assayed for cleavage and polyadenylation in vitro. Although they appeared similarly active in cleavage as a TAP-Fip1p wild-type CPF (Figure 2B, compare lane 6 and 7 with lane 2), they failed to polyadenylate the CYC1pre substrate unless recombinant poly(A) polymerase (Pap1p) complemented them in the assays (Figure 2C, compare lanes 2 and 3, and 7 and 8). In comparison, TAP-Fip1p complexes purified from the wild-type or mutant pti1-2 or ref2-2 strains were proficient on their own (Figure 2C, lanes 4–6 and lane 9). The inefficient polyadenylation activity could be explained at least in part by the very low levels of Pap1p detected by western blotting of TAP-Ref2p and Pti1p-TAP complexes compared with all three TAP-Fip1p complexes (Figure 2D). These results showed that TAP-Ref2p and Pti1p-TAP factors could associate with CF I in a complex that could at least specifically cleave an mRNA precursor. However, Pti1p and Ref2p essential function did not seem to be related to 3′ end formation of this class of RNA precursors. As pol II also transcribes precursors of small RNAs, we investigated whether mutations in PTI1 and REF2 would affect snoRNA 3′ end maturation. snoRNA 3′ end formation is impaired in pti1 and ref2 mutants In yeast, snoRNAs are transcribed by pol II, in most cases as independent, either mono- or polycistronic units. In some instances, the 5′ end of the snoRNA corresponds to the 5′ end of the primary transcript and carries a trimethylguanosine cap (Chanfreau et al., 1998a; Fatica et al., 2000). However, it also happens that the 5′ end is matured by endonucleolytic cleavage followed by exonucleolytic trimming. Likewise, the 3′ end is formed by exonucleolytic degradation of the precursor after cleavage. The entry sites for exonucleases are generated by the RNA endonuclease Rnt1p in the 5′ portion of the precursor, but this is very often not the case for the 3′ end (Chanfreau et al., 1998a; Allmang et al., 1999; van Hoof et al., 2000). Recent studies implicated CF IA but not CPF as essential for 3′ end cleavage of small stable RNAs based on the analysis of several mutants in both factors (Fatica et al., 2000; Morlando et al., 2002). To test whether pti1-2 and ref2-2 mutations may affect 3′ end formation of snoRNAs, we performed primer extension analysis of transcripts from the SNR13-TRS31 region of the genome as described previously (Figure 3A; Steinmetz et al., 2001; Morlando et al., 2002). An oligonucleotide complementary to the TRS31 ORF revealed extended transcripts by reverse transcription of total RNA extracted from an rna15 mutant strain after shift to 37°C (Figure 3B, lanes 10–12, product B) but not from the wild-type strain (Figure 3B, lanes 1–3, product A). This indicated that Rna15p was involved in vivo in box C/D snR13 3′ end formation as described (Morlando et al., 2002). Interestingly, the strong rna15-1 mutant allele tested here was impaired already at permissive temperature (24°C; Figure 3B, lane 10). More strikingly, the pti1-2 strain showed a rapid and dramatic accumulation of extended product B after shift to the restrictive temperature (Figure 3B, lanes 4–6). Likewise, the ref2-2 strain exhibited product B accumulation although to a lesser extent (Figure 3B, lanes 7–9). In the pti1-2 mutant only, product A was undetectable after shift to 37°C, suggesting that initiation of transcription at the TRS31 promoter was almost entirely abolished by promoter occlusion (Greger et al., 2000). Unlike with pti1-2 and ref2-2, extended product B did not accumulate in the rna15-1 mutant, which is also impaired in mRNA 3′ end formation, probably because the TRS31 mRNA was not polyadenylated properly and was thus degraded (Figure 3B). Figure 3.Reverse transcription analysis of extended transcripts of some snoRNAs in wild-type and mutant strains. (A) Schematic representation of the SNR13-TRS31 region of the chromosome. 'A' refers to reverse transcription products in a wild-type strain, and 'B' to snR13 3′-extended transcripts. (B) Polyacrylamide gel electrophoresis of reverse transcripts from wild-type (WT), pti1-2, ref2-2 and rna15-1 strains 0, 1 and 2 h after shift to 37°C. 'A' and 'B' are as depicted in (A); M, molecular weight markers in number of nucleotides. (C) Reverse transcription analysis of several box C/D and H/ACA snoRNAs in wild-type and pti1-2 and ref2-2 mutant strains. The pol III-transcribed U6 RNA is given as a normalization control for the reverse transcription. Download figure Download PowerPoint Several other snoRNAs, including box C/D snR71, snR4, snR50 and snR45, and box H/ACA snR33, snR189, snR3, snR9 and snR32, also showed readthrough transcripts in pti1-2 and ref2-2 mutants (Figure 3C). Surprisingly, ref2-2 mutant reproducibly displayed a more pronounced effect on the accumulation of the 3′-extended forms of box H/ACA snR33 and snR189 than that observed with the pti1-2 mutant, whereas snR32 3′ end formation looked equally affected by both mutations. The reason for these differences currently is unclear, but this suggests that Pti1p and Ref2p have no redundant function in snoRNA 3′ end formation. Therefore, CPF-associated Pti1p and Ref2p are probably generally required for 3′ end formation of independently transcribed snoRNAs. Overexpression of Pti1p uncouples cleavage and polyadenylation Our results suggested that the same global protein machinery, i.e. CF I + CPF, is required for pol II-transcribed polyadenylated and non-polyadenylated RNA 3′ end maturation. Since polyadenylation of snoRNAs is detrimental to their function (Fatica et al., 2000), this apparatus must thus discriminate between mRNA and snoRNA precursors. This may be achieved in part through recognition of cis-acting elements specific for both precursors (Fatica et al., 2000; Edmonds, 2002). However, trans-acting factors might be essential to mediate uncoupling of cleavage and polyadenylation. To address whether Pti1p, which generally exhibited the strongest snoRNA 3′ end formation defect in its mutant form, might play such a role, we constructed a strain where expression of Pti1p is driven by the strong inducible/repressible GAL10 promoter in order to intensify its putative function in uncoupling the two reactions. Our construct also introduced a ubiquitin moiety at the N-terminus such that the protein started with the destabilizing amino acid arginine after in vivo de-ubiquitylation and therefore was rapidly degraded (Figure 4A; Billy et al., 2000). Extracts prepared from cells grown in galactose (GAL) or after a shift to a glucose-containing medium (GLU) for 4 h were assayed in vitro for pre-mRNA 3′ end processing. Cleavage of the CYC1 precursor was normal with both the GAL and GLU extracts (Figure 4B, lanes 2 and 3). However, polyadenylation of the CYC1pre transcript was significantly impaired when Pti1p was overexpressed in galactose, whereas it became normal as Pti1p expression was repressed in glucose (Figure 4B, compare lanes 5 and 6). As a control, an extract prepared from a wild-type, unmodified strain grown in galactose could process the CYC1 precursors efficiently (Figure 4B, lanes 7–10). A western blot analysis of the GAL and GLU extracts showed that Pti1p was undetectable in glucose-grown cells, whereas levels of the CF IA component Rna15p or CPF subunits such as Fip1p and even the poly(A) polymerase Pap1p were not affected by the ubiquitin system in either growth condition (Figure 4C). Depletion of Pti1p in vivo correlated with the same 3′ end formation defect as observed with the temperature-sensitive allele pti1-2 (Figure 4D, lanes 1–5). The inhibitory effect of Pti1p overexpression on polyadenylation was also examined with an extract prepared from a strain where the destabilizing ubiquitin-arginine conditional system was omitted in order to avoid possible side effects of the method (Figure 5A; Lafontaine and Tollervey, 1996). In this situation, Pti1p overexpression affected polyadenylation even more dramatically (Figure 5B, lanes 5 and 6) whereas cleavage remained unaffected (Figure 5B, lanes 2 and 3). Low levels of Pti1p were still visible on western blots in the GLU extracts but nevertheless allowed polyadenylation to proceed (Figure 5C). Here again, neither CF IA subunit Rna15p nor CPF-associated Pap1p levels declined after the glucose shift (Figure 5C). It is worth recalling here that other CPF subunits subjected to identical conditional expression have been reported to show opposite effects, i.e. inhibition of polyadenylation following repression of protein expression (e.g. Ysh1p and Yth1p; Jenny et al., 1996; Barabino et al., 2000). Together with the data obtained with TAP-Ref2p and Pti1p-TAP complexes on polyadenylation (Figure 2C), these results suggested that Pti1p, and probably Ref2p, may function in snoRNA 3′ end formation by uncoupling cleavage and polyadenylation. Figure 4.Conditional expression of PTI1 affects 3′ end formation of pol II-transcribed RNAs. (A) A destabilizing ubiquitin-arginine moiety was fused to Pti1p and the hybrid construct was expressed under the control of the inducible GAL10 promoter. The fusion was reintroduced at the PTI1 chromosomal locus (Billy et al., 2000). (B) Extracts prepared from GAL::R-Pti1p-expressing cells grown in galactose (GAL) or after a 4 h shift to glucose (GLU) were tested for cleavage of the CYC1 precursor (lanes 1–3) and polyadenylation of the CYC1pre RNA (lanes 4–6). A wild-type extract made from an unmodified BMA64 wild-type strain grown in galactose was tested for cleavage (lanes 7 and 8) and polyadenylation (lanes 9 and 10) as above. (C) Immunoblot analysis of the GAL and GLU extracts (20 μg) with polyclonal antibodies to Pti1p (1:500), Rna15p (1:10 000), Fip1p (1:10 000) and Pap1p (1:2000). The arrowheads point to irrelevant polypeptides that cross-react with antibodies to Pti1p and Rna15p. (D) Analysis of snR13 extended transcripts in the wild-type, pti1-2 and GAL::R-Pti1p conditional mutant strains. The reverse transcription was performed on RNAs extracted from wild-type and pti1-2 cells after a shift to 37°C for 2 h (lanes 1 and 2, respectively), or from GAL::R-Pti1p-expressing cells grown in galactose (lane 3, GAL t = 0) then re-inoculated in either galactose or glucose medium for 4 h (lanes 4 and 5, respectively). 'B' refers to snR13 extended transcripts as diagrammed in Figure 3A. U6 serves as a normalization control for the reverse transcription. M, molecular weight markers in number of nucleotides. Download figure Download PowerPoint Figure 5.Conditional expression of Ptip1 under the GAL promoter. (A) Schematic representation of the hybrid construct introduced at the PTI1 chromosomal locus (Lafontaine and Tollervey, 1996). The Pti1 protein is tagged at the N-terminus with the c-myc epitope. Its expression is induced in galactose-containing medium and repressed in glucose. The black box represents the c-myc epitope tag. (B) Cleavage and polyadenylation assays were performed with extracts prepared with the GAL::(c-myc)-Pti1p-expressing strain as in Figure 4B. (C) Immunoblot a
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