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• W1979632388 abstract "Regulation of poly(A) tail length during mRNA 3′-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3′-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3′-end formation were purified from wild-type and pan mutant cells. In both situations, 3′-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast. Regulation of poly(A) tail length during mRNA 3′-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3′-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3′-end formation were purified from wild-type and pan mutant cells. In both situations, 3′-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast. Eukaryotic mRNA 3′-end formation is a two-step reaction (reviewed in Refs. 1Edmonds M. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 285-389Crossref PubMed Google Scholar, 2Zhao J. Hyman L. Moore C. Microbiol. Mol. Biol. Rev. 1999; 63: 405-445Crossref PubMed Google Scholar, 3Wahle E. Rüegsegger U. FEMS Microbiol. Rev. 1999; 23: 277-295Crossref PubMed Google Scholar). The RNA polymerase II-transcribed mRNA precursor is first cleaved in its 3′-untranslated region and the upstream fragment is subsequently polyadenylated. The mechanism and the machinery involved in this processing have been well conserved through evolution. Human and yeast share similarities in the reaction mechanism and in the composition of the implicated machinery. In mammals, the cleavage reaction can be recapitulated in vitro in the presence of cleavage factors I and II (CF Im and CF IIm), 1The abbreviations used are: CF Im and CF IIm, mammalian cleavage factors I and II; CPSF, cleavage and polyadenylation specificity factor; PAP, poly(A) polymerase (mammalian); PABPN1, mammalian nuclear poly(A)-binding protein 1; Pab1p, yeast poly(A)-binding protein 1; CF IA and CF IB, yeast cleavage factors IA and IB; PAN, yeast Pab1p-dependent poly(A) nuclease; TAP, tandem affinity purification; CYC1, iso-1-cytochrome c precursor RNA; nt, nucleotide. the cleavage stimulation factor (CstF), the cleavage and polyadenylation specificity factor (CPSF), and the poly(A) polymerase (PAP). Controlled poly(A) tail synthesis requires PAP, CPSF, and the nuclear poly(A)-binding protein 1 (PABPN1) (4Rüegsegger U. Blank D. Keller W. Mol. Cell. 1998; 1: 243-253Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 5Wahle E. J. Biol. Chem. 1995; 270: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Mammalian poly(A) tails are synthesized to an average length of 250 adenosine residues. PAP alone does not have any pronounced specificity with regard to the RNA substrate and is poorly active on its own. Its activity is stimulated by two factors, CPSF, which binds to the highly conserved element AAUAAA, and PABPN1, which interacts with the growing poly(A) tail. The combined activity of both stimulatory factors leads to fully processive elongation such that a complete poly(A) tail can be synthesized without dissociation of the polymerase. Once poly(A) tails have reached ∼250 nucleotides, elongation terminates by switching from a processive to a slower distributive mode. Although the exact mechanism that controls poly(A) tail length is unclear, one possibility is that once 250 adenosine residues have been polymerized, disruption of the polyadenylation complex PAP-CPSF-PABPN1 takes place perhaps because the tail is too long to mediate interactions between the three components of the complex (5Wahle E. J. Biol. Chem. 1995; 270: 2800-2808Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6Kerwitz Y. Kuhn U. Lilie H. Knoth A. Scheuermann T. Friedrich H. Schwarz E. Wahle E. EMBO J. 2003; 22: 3705-3714Crossref PubMed Scopus (124) Google Scholar). Poly(A) tail length control in S. cerevisiae is poorly understood, and there is a controversy about which factors are required. In yeast, poly(A) tails are shorter with an average length of about 70 adenosine residues. The two steps of mRNA 3′-end formation require the cleavage factors IA (CF IA) and IB (CF IB or Nab4p/Hrp1p) and the cleavage and polyadenylation factor (CPF) containing the poly(A) polymerase (Pap1p), which is dispensable for the cleavage step of the reaction (7Minvielle-Sebastia L. Beyer K. Krecic A.M. Hector R.E. Swanson M.S. Keller W. EMBO J. 1998; 17: 7454-7468Crossref PubMed Scopus (89) Google Scholar). Poly(A) tail length control can be reconstituted in vitro in the presence of these three factors and a poly(A)-binding protein. It was initially suggested that the major cytoplasmic poly(A)-binding protein Pab1p was responsible for the poly(A) tail length control during de novo synthesis by inhibiting Pap1p activity (8Lingner J. Radkte I. Wahle E. Keller W. J. Biol. Chem. 1991; 266: 8741-8746Abstract Full Text PDF PubMed Google Scholar, 9Minvielle-Sebastia L. Preker P.J. Wiederkehr T. Strahm Y. Keller W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7897-7902Crossref PubMed Scopus (142) Google Scholar, 10Amrani N. Minet M. Le Gouar M. Lacroute F. Wyers F. Mol. Cell. Biol. 1997; 17: 3694-3701Crossref PubMed Scopus (120) Google Scholar). This hypothesis was supported by several observations. First, Pab1p co-purifies with CF IA and specifically interacts with the Rna15p subunit using either two-hybrid analysis or co-immunoprecipitation and PAB1 is a multicopy suppressor of rna15–2, coding for a subunit of CF IA. Second, poly(A) tails are at least 50 nt longer in vitro when the mRNA precursor is incubated in the pab1Δ mutant extracts. Third, complementation with recombinant Pab1p restores normal length control to the pab1Δ extracts but also induces a shortening of the transcript (9Minvielle-Sebastia L. Preker P.J. Wiederkehr T. Strahm Y. Keller W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7897-7902Crossref PubMed Scopus (142) Google Scholar, 10Amrani N. Minet M. Le Gouar M. Lacroute F. Wyers F. Mol. Cell. Biol. 1997; 17: 3694-3701Crossref PubMed Scopus (120) Google Scholar). It has been suggested that Pab1p acts by stimulating the poly(A) nuclease PAN, which might balance excessive growth of poly(A) tails by trimming them from the 3′-end (11Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (182) Google Scholar). Deadenylation is the first step in the general mRNA turnover in eukaryotes (reviewed in Ref. 12Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (652) Google Scholar). In yeast, two different complexes have been identified as mRNA deadenylases. The predominant one is the conserved Ccr4p/Pop2p (or Caf1p) complex, which is inhibited by Pab1p. PAN is the other one, which is responsible for the residual deadenylation in a ccr4Δ mutant (13Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). PAN is composed of two subunits, Pan2p (127 kDa) and Pan3p (76 kDa), which are essential for its nuclease activity (14Boeck R. Tarun S. Rieger M. Deardorff J.A. Müller-Auer S. Sachs A.B. J. Biol. Chem. 1996; 271: 432-438Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 15Brown C.E. Tarun S.Z. Boeck R. Sachs A.B. Mol. Cell. Biol. 1996; 16: 5744-5753Crossref PubMed Scopus (130) Google Scholar). Pan2p is the catalytic subunit and is a member of the RNaseD superfamily, while Pan3p is a positive regulator of PAN activity. Previously, poly(A) tail length control was thought to involve PAN in an mRNA-dependent manner (11Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (182) Google Scholar). More recently, the PAN complex was suggested to be a component of the nuclear pre-mRNA 3′-end processing machinery together with CF IA, CF IB, and CPF (16Mangus D.A. Evans M.C. Agrin N.S. Smith M. Gongidi P. Jacobson A. Mol. Cell. Biol. 2004; 24: 5521-5533Crossref PubMed Scopus (68) Google Scholar, 17Mangus D.A. Smith M.M. McSweeney J.M. Jacobson A. Mol. Cell. Biol. 2004; 24: 4196-4206Crossref PubMed Scopus (47) Google Scholar). We have shown previously that the nuclear poly(A)-binding protein Nab2p specifically controls poly(A) tail length in vivo and in vitro in yeast (18Hector R.E. Nykamp K.R. Dheur S. Anderson J.T. Non P.J. Urbinati C.R. Wilson S.M. Minvielle-Sebastia L. Swanson M.S. EMBO J. 2002; 21: 1800-1810Crossref PubMed Scopus (141) Google Scholar). In this report, we investigated whether PAN might play an additional role in de novo poly(A) tail synthesis in vitro. Yeast Strains—The pan2Δ pan3Δ strain (YAS1944) has been described elsewhere (15Brown C.E. Tarun S.Z. Boeck R. Sachs A.B. Mol. Cell. Biol. 1996; 16: 5744-5753Crossref PubMed Scopus (130) Google Scholar). The pab1 deletion is rescued by deletion of RPL46 in the strain YAS394 (19Sachs A. Deardorff J.A. Cell. 1992; 70: 961-973Abstract Full Text PDF PubMed Scopus (160) Google Scholar). Strains expressing the C-terminally tandem affinity purification (TAP)-tagged Pan2p (YSD7) or Pan3p (YSD8) were constructed in BMA64 (ade2–1 leu2–3,112 ura3–1 trp1Δ his3–11,15 can1–100) (20Baudin-Baillieu A. Guillemet E. Cullin C. Lacroute F. Yeast. 1997; 13: 353-356Crossref PubMed Scopus (55) Google Scholar) according to Rigaut et al. (21Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). Rna15p and Fip1p were N-terminally TAP-tagged either in the wild-type strain BMA64 (YSD12 and YSD10, respectively) or in the pan2Δ pan3Δ strain YAS1944 (YSD9 and YSD11, respectively) according to Dheur et al. 2S. Dheur, F. Voisinet-Hakil, L. Minvielle-Sebastia, manuscript in preparation. This N-terminal TAP-tagging method was performed by integrating at the chromosomal locus using a cassette which adds a TAP-tag identical to that described previously (21Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). Strains expressing both N-terminally TAP-tagged Rna15p and Fip1p were also constructed in either the wild-type BMA64 (YSD13) or the pan2Δ pan3Δ strain YAS1944 (YSD14). In Vitro 3′-End Processing Assays—Polyadenylation-competent extracts were prepared using a spheroplast procedure (22Butler J.S. Sadhale P.P. Platt T. Mol. Cell. Biol. 1990; 10: 2599-2605Crossref PubMed Google Scholar). Tandem affinity purification of CF IA and CPF was performed according to Rigaut et al. (21Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). Pre-mRNA cleavage and polyadenylation were assayed essentially as previously described (7Minvielle-Sebastia L. Beyer K. Krecic A.M. Hector R.E. Swanson M.S. Keller W. EMBO J. 1998; 17: 7454-7468Crossref PubMed Scopus (89) Google Scholar, 23Minvielle-Sebastia L. Preker P.J. Keller W. Science. 1994; 266: 1702-1705Crossref PubMed Scopus (141) Google Scholar). Briefly, polyadenylation in cell extracts was assayed at 30 °C with 20 μg of extract and stopped at 30, 60, or 90 min by addition of proteinase K. For reconstitution assays with purified factors, reactions contained either 1 μl of CF IA (TAP-Rna15p) + 1 μl of CPF (TAP-Fip1p) or 2 μl of [CF IA + CPF] (TAP-Rna15p and TAP-Fip1p) supplemented with 20 ng of recombinant Nab4p (7Minvielle-Sebastia L. Beyer K. Krecic A.M. Hector R.E. Swanson M.S. Keller W. EMBO J. 1998; 17: 7454-7468Crossref PubMed Scopus (89) Google Scholar) and either 25–400 ng of recombinant Pab1p (kind gift of Alan Sachs) or 40–120 ng of recombinant Nab2p (18Hector R.E. Nykamp K.R. Dheur S. Anderson J.T. Non P.J. Urbinati C.R. Wilson S.M. Minvielle-Sebastia L. Swanson M.S. EMBO J. 2002; 21: 1800-1810Crossref PubMed Scopus (141) Google Scholar). For the time course experiment, a 10-fold reaction mixture containing 20 μl of [CF IA + CPF] and 200 ng of recombinant Nab4p was supplemented with either 2 μg of recombinant Pab1p or 1.2 μg of recombinant Nab2p. This mixture was preincubated at 30 °C for 15 min, and polyadenylation was initiated by addition of labeled transcript. Aliquots were withdrawn at indicated times and the reaction was stopped and analyzed on a 40 cm long 6% polyacrylamide-8.3 m urea gel. For the inactivation of Nab2p by Kap104p experiment, 0.5 μg of Nab2p was preincubated with increasing amounts of Kap104p (0.5, 1, 1.5, and 2 μg) for 15 min at 30 °C in the buffer used for the 3′-end processing assays. Recombinant Kap104p was prepared as described (24Lee D.C. Aitchison J.D. J. Biol. Chem. 1999; 274: 29031-29037Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). After incubation, the recombinant proteins were added to standard processing reactions and performed as described above. Co-immunopurification and Immunoblotting—For immunodepletion analysis using the pab1Δ (YAS394) polyadenylation-competent extracts, 200 μg of extract were incubated with either anti-Nab2p 3F2, anti-Kap104p 1D12, anti-Nab4p 3H1, or anti-Pub1p 4C3 monoclonal antibodies prebound to rabbit anti-mouse antibodies coupled to protein A-Sepharose beads. Immunopurification was performed for 30 min at 4 °C in 25 μl of a buffer containing 20 mm Hepes-KOH, pH 7.0, 0.2 mm EDTA, 50 mm KCl, 20% glycerol, 0.5 mm dithiothreitol, 0.01% Nonidet P-40, 0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 1 μg/ml chymostatin, 1 mm ϵ-aminocaproic acid, 1 mm ρ-aminobenzamidine, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 2 mm Pefabloc. Aliquots of the supernatant were analyzed by immunoblotting as described (25Anderson J.T. Wilson S.M. Datar K.V. Swanson M.S. Mol. Cell. Biol. 1993; 13: 2730-2741Crossref PubMed Scopus (158) Google Scholar), using a 1:500 dilution of anti-Nab2p 3F2 or anti-Kap104p 1D12 antibodies and a 1:5000 dilution of horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (Amersham Biosciences). Cellular Immunofluorescence—The subcellular distributions of TAP-tagged Pan2p, Pan3p, and Rna15p were examined by cell immunofluorescence as described previously (18Hector R.E. Nykamp K.R. Dheur S. Anderson J.T. Non P.J. Urbinati C.R. Wilson S.M. Minvielle-Sebastia L. Swanson M.S. EMBO J. 2002; 21: 1800-1810Crossref PubMed Scopus (141) Google Scholar) using an Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Molecular Probes, Eugene, OR). Cells were visualized using a Zeiss Axioskop 2 mot plus microscope equipped with a ×100 fluorescence/differential contrast objective and images were captured with a Zeiss monochrome digital camera. In Vitro Synthesized mRNA Poly(A) Tails Are Not Aberrantly Long in PAN-deficient Cell Extracts Compared with Pab1p-deficient Extracts—To evaluate the putative involvement of the PAN nuclease during in vitro poly(A) tail synthesis, we performed a time course experiment and characterized the 3′-end processing activity of extracts prepared from either the wild-type, the pan2Δ pan3Δ, or the pab1Δ mutant strains. The 3′-untranslated region of the mRNA precursor CYC1 was transcribed in vitro in the presence of a radiolabeled nucleotide and then incubated with the different yeast extracts. As shown in Fig. 1, the CYC1 precursor (lane 1, shown as a gray and white rectangle and running with the 309-nt marker) was cleaved at the normal poly(A) site. The resulting upstream fragment (running between the 180- and 190-nt marker bands and represented as a gray box) was polyadenylated in wild-type extracts. This polyadenylation reaction resulted in poly(A) tails with an average length of 60 adenosine residues, which ran above the upstream fragment in the region of the 242-nt marker (lanes 2 and 3, gray box followed by AAAA). Subsequently, poly(A) tails were shortened (Fig. 1, lane 4). This time course assay could reflect an equilibrium between synthesis and degradation that occurs within the first hour, which is followed by a net deadenylation because of a reduced polyadenylation rate. Alternatively, this experiment could highlight a two-step reaction in which trimming would be the final processing step that follows polymerization. Fig. 1 shows that the PAN-deficient extracts displayed a polyadenylation activity that was not dramatically different from that observed with wild-type extracts (Fig. 1, compare lanes 5–7 with lanes 2–4). Although cleavage activity was less efficient than that of the wild-type extract, this result should be interpreted with caution, since cleavage efficiency is highly dependent upon extract preparation and differs from one extract to another with no obvious correlation to the strain used. 3S. Dheur and L. Minvielle-Sebastia, unpublished observation. Poly(A) tails in the pan mutant extracts were slightly longer, but their length was rather constant with time, which is the most striking difference with the wild-type extracts (Fig. 1, compare lane 4 with lane 7). On the other hand, the pab1 mutant extracts were noticeably more affected in poly(A) tail length control (Fig. 1, lanes 8–10). In this case, the average tail length was ∼50 nt longer, although very long tails were detectable. Because of this increasing length, the polyadenylated product overlapped with the precursor in lanes 8–10 of Fig. 1. Tail length also remained constant with time. Altogether, these results suggested that Pab1p restricts poly(A) tail length only to a certain extent in vitro and that the Pab1p-dependent nuclease PAN does not play a direct role in de novo poly(A) tail synthesis but might be involved in a trimming activity that occurs following polyadenylation. However, several observations argue against a role for Pab1p in poly(A) tail length control in vivo. First, Pab1p is an abundant cytoplasmic protein that is very weakly detected in the nucleus (25Anderson J.T. Wilson S.M. Datar K.V. Swanson M.S. Mol. Cell. Biol. 1993; 13: 2730-2741Crossref PubMed Scopus (158) Google Scholar). Second, Pab1p is a major contaminant of the complexes purified from yeast. For instance, it was present in 10% of the tandem affinity purified complexes in the large scale approach reported in Gavin et al. (26Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google Scholar). Third, the very long tails observed in vitro with pab1 mutant extracts do not reflect the in vivo situation where tails are only 3–20 nt longer in the absence of Pab1p (27Sachs A.B. Davis R.W. Cell. 1989; 58: 857-867Abstract Full Text PDF PubMed Scopus (392) Google Scholar). These arguments led us to consider the possibility that the poly(A) tail length regulatory factor in vivo might have been lost or inactivated during extract preparation. The Poly(A) Tail Length Regulatory Function of Nab2p in Vitro Might Be Obscured because of Its Binding to the Nuclear Transport Factor Kap104p during Extract Preparation—We recently provided evidence that the hnRNP Nab2p was another candidate for controlling mRNA poly(A) tail synthesis in vivo and in vitro (18Hector R.E. Nykamp K.R. Dheur S. Anderson J.T. Non P.J. Urbinati C.R. Wilson S.M. Minvielle-Sebastia L. Swanson M.S. EMBO J. 2002; 21: 1800-1810Crossref PubMed Scopus (141) Google Scholar). Unlike Pab1p, Nab2p is a predominantly nuclear protein (25Anderson J.T. Wilson S.M. Datar K.V. Swanson M.S. Mol. Cell. Biol. 1993; 13: 2730-2741Crossref PubMed Scopus (158) Google Scholar, 28Wilson S.M. Datar K.V. Paddy M.R. Swedlow J.R. Swanson M.S. J. Cell Biol. 1994; 127: 1173-1184Crossref PubMed Scopus (111) Google Scholar) that plays a dual role in the termination of mRNA polyadenylation and nuclear export. Nab2p is a shuttling protein that is reimported to the nucleus by way of its association with the karyopherin Kap104p (24Lee D.C. Aitchison J.D. J. Biol. Chem. 1999; 274: 29031-29037Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 29Aitchison J.D. Blobel G. Rout M.P. Science. 1996; 274: 624-627Crossref PubMed Scopus (271) Google Scholar). Kap104p and Nab2p form a complex in the cytosol and binding of Kap104p to Nab2p destabilizes Nab2p-RNA interactions (24Lee D.C. Aitchison J.D. J. Biol. Chem. 1999; 274: 29031-29037Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Because the poly(A) tail regulatory activity of Nab2p is dependent on its RNA-binding properties, we speculated that Kap104p binds to free Nab2p in extracts resulting in an inhibition of its poly(A) tail regulatory activity. However, the abundant poly(A)-binding protein Pab1p masked the loss of active Nab2p in extracts as it could substitute for it in polyadenylation assays (9Minvielle-Sebastia L. Preker P.J. Wiederkehr T. Strahm Y. Keller W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7897-7902Crossref PubMed Scopus (142) Google Scholar, 10Amrani N. Minet M. Le Gouar M. Lacroute F. Wyers F. Mol. Cell. Biol. 1997; 17: 3694-3701Crossref PubMed Scopus (120) Google Scholar). As a result, the loss of Nab2p activity during extract preparation would therefore explain the complete absence of poly(A) tail length control in pab1Δ extracts. To test this hypothesis, we determined whether the anti-Kap104p monoclonal antibody 1D12 (mAb 1D12) could immunodeplete Nab2p from 3′-end processing extracts. In this assay, pab1Δ extracts were immunodepleted with several antibodies, and the presence of Nab2p and Kap104p in the antigen-depleted supernatants was analyzed by Western blot (see “Experimental Procedures”) (Fig. 2). As anticipated, the anti-Kap104p antibody not only depleted the extracts of Kap104p but also the majority of Nab2p (Fig. 2, lane 3). It also appeared that Kap104p was considerably more abundant than Nab2p, since the anti-Nab2p antibody failed to completely immunodeplete Kap104p from the extracts (Fig. 2, lane 4). As Nab4p is also reimported by Kap104p to the nucleus (24Lee D.C. Aitchison J.D. J. Biol. Chem. 1999; 274: 29031-29037Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), this might correspond to a Kap104p-Nab4p complex as immunodepletion with antibodies directed against Nab4p failed to pull down all Kap104p as well (Fig. 2, lane 5). Neither the anti-Nab4p antibody nor an antibody against the unrelated protein Pub1p led to Nab2p depletion in the extracts (Fig. 2, lanes 5 and 6, respectively, compared with the mock depleted extract in lane 2). These results suggested that Kap104p binds Nab2p in polyadenylation-competent extracts. This might explain why control of poly(A) tail synthesis was lost in vitro in the pab1Δ extracts, which does not reflect the in vivo situation in the pab1Δ strain (27Sachs A.B. Davis R.W. Cell. 1989; 58: 857-867Abstract Full Text PDF PubMed Scopus (392) Google Scholar). This data supported our previous proposal that Nab2p is the bona fide poly(A) tail length regulatory factor in yeast and that its inactivation by Kap104p was masked by the activity of the abundant poly(A)-binding protein Pab1p in 3′-end processing extracts. The Absence of PAN Does Not Affect the Composition of Purified CF IA and CPF Complexes—If PAN functions in poly(A) tail length control during de novo synthesis, reconstitution of regulated mRNA polyadenylation with 3′-end processing factors purified from extracts lacking this deadenylase should be compromised. Therefore, we purified CF IA and CPF from a pan2Δ pan3Δ strain to recapitulate the pre-mRNA 3′-end processing reaction in vitro in the absence of PAN activity. The two complexes were purified either separately or together from a pan mutant and a wild-type strain using the TAP method (21Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar). CF IA and CPF were purified from strains expressing N-terminally TAP-tagged Rna15p (for CF IA purification) and/or TAP-tagged Fip1p (for CPF purification). Purified factors were visualized on silver-stained polyacrylamide gels (Fig. 3). The majority of the expected bands corresponding to already identified subunits could be detected (26Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google Scholar), and no significant difference in the composition of the factors could be found between the wild-type and the pan mutant backgrounds. Normal Cleavage and Polyadenylation of Pre-mRNA Can Be Recapitulated in Vitro with Purified Factors in the Absence of PAN—To evaluate the effect of PAN absence on poly(A) tail formation, we performed in vitro assays with the TAP-purified CF IA and CPF. Their activities were tested in vitro for pre-mRNA cleavage and polyadenylation in combination with recombinant Nab4p (CF IB) and increasing amounts of either recombinant Pab1p or Nab2p (Fig. 4). In the presence of CF IA, CF IB, and CPF, the 3′-untranslated region of CYC1 precursor was cleaved and hyperadenylated (Fig. 4, lanes 2 and 8). In these conditions, poly(A) tails were polymerized to a length exceeding 400 nt. Addition of increasing amounts of Pab1p reduced the average tail length to a size similar to that obtained with wild-type cell extracts (Fig. 4, compare lanes 3–7 and 9–13 with lane 14). However, two major bands (under the precursor) and very long tails (running above the precursor and higher than the 622-nt marker) could be observed with high amounts of Pab1p under conditions where cleavage began to be inhibited (Fig. 4, lanes 6 and 7 and lanes 12 and 13). The two bands were not detected in previous studies where shorter polyacrylamide gels with lower resolution were used. These two major polyadenylated products that appeared with high amounts of Pab1p (Fig. 4, lanes 12 and 13) could have been interpreted as steps in poly(A) degradation. However, it was not dependent on PAN activity, since these bands were also present with the factors purified from panΔ extracts (Fig. 4, lanes 6 and 7). In general, no significant differences were observed between wild-type and panΔ factor activities (Fig. 4, compare lanes 2–7 with lanes 8–13). Addition of increasing amounts of Nab2p also resulted in a decrease of poly(A) tail length (Fig. 4, lanes 16–20 and 21–25). In contrast to Pab1p, we could observe a smear corresponding to the mature polyadenylated product below the precursor. In addition, the hyperadenylated species running above the precursor were no longer visible (Fig. 4, lanes 20 and 25). The reaction appeared to be more efficient than with Pab1p, since as little as 80 ng of Nab2p could suppress the formation of hyperadenylated products, which differs from the situation found with Pab1p. Noticeably, wild-type and panΔ-purified factors displayed the same activity (Fig. 4, compare lanes 16–20 with lanes 21–25). It is important to mention that the lower levels of the CYC1 precursor remaining" @default.
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• W1979632388 title "Yeast mRNA Poly(A) Tail Length Control Can Be Reconstituted in Vitro in the Absence of Pab1p-dependent Poly(A) Nuclease Activity" @default.
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