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• W2071720275 abstract "Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3′-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation. Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3′-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation. The cap structure and the poly(A) tail are the two boundary marks that define the extreme borders of a eukaryotic mRNA (1.Shatkin A.J. Manley J.L. Nat. Struct. Biol. 2000; 7: 838-842Crossref PubMed Scopus (254) Google Scholar). Both elements play several important roles in regulating eukaryotic gene expression, and in particular, they influence the fate of mRNA, including its synthesis, maturation, translation, and stability (reviewed in Refs. 1.Shatkin A.J. Manley J.L. Nat. Struct. Biol. 2000; 7: 838-842Crossref PubMed Scopus (254) Google Scholar, 2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar, 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar, 4.Sachs A.B. Varani G. Nat. Struct. Biol. 2000; 7: 356-361Crossref PubMed Scopus (104) Google Scholar, 5.Wilusz C.J. Wormington M. Peltz S.W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (628) Google Scholar). A number of proteins that change dynamically during the mRNA life cycle interact with each of the elements, and it has become evident during recent years that these proteins play critical roles in regulating processes that are influenced by or dependent on the two elements (reviewed in Refs. 1.Shatkin A.J. Manley J.L. Nat. Struct. Biol. 2000; 7: 838-842Crossref PubMed Scopus (254) Google Scholar, 2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar, 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar, 4.Sachs A.B. Varani G. Nat. Struct. Biol. 2000; 7: 356-361Crossref PubMed Scopus (104) Google Scholar, 5.Wilusz C.J. Wormington M. Peltz S.W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (628) Google Scholar, 6.Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell. Biol. 2002; 3: 195-205Crossref PubMed Scopus (1119) Google Scholar). In the mammalian cell nucleus, the cap is recognized by the nuclear cap binding complex (CBC) 4The abbreviations used are: CBC, cap binding complex; PARN, poly(A)-specific ribonuclease; PABPC, cytoplasmic poly(A)-binding protein; eIF, eukaryotic translation initiation factor; ARE, AU-rich elements; NMD, nonsense-mediated decay.4The abbreviations used are: CBC, cap binding complex; PARN, poly(A)-specific ribonuclease; PABPC, cytoplasmic poly(A)-binding protein; eIF, eukaryotic translation initiation factor; ARE, AU-rich elements; NMD, nonsense-mediated decay. (7.Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar), and the poly(A) tail is associated with nuclear poly(A)-binding protein 1 (PABPN1) (8.Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (257) Google Scholar). CBC is a heterodimeric complex that consists of a small (CBP20) and a large (CBP80) protein subunit and plays direct roles in pre-mRNA splicing (7.Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar), 3′ end formation (9.Flaherty S.M. Fortes P. Izaurralde E. Mattaj I.W. Gilmartin G.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11893-11898Crossref PubMed Scopus (173) Google Scholar), and uridylic acid-rich small nuclear RNA export (10.Visa N. Izaurralde E. Ferreira J. Daneholt B. Mattaj I.W. J. Cell Biol. 1996; 133: 5-14Crossref PubMed Scopus (196) Google Scholar). In the cytoplasm, CBC is replaced by the cytoplasmic cap-binding protein, also known as eukaryotic translation initiation factor 4E (eIF4E), which together with factors eIF4A and eIF4G is responsible for initiation of cap-dependent mRNA translation (see Refs. 11.Ishigaki Y. Li X. Serin G. Maquat L.E. Cell. 2001; 106: 607-617Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar and 12.Lejeune F. Ranganathan A.C. Maquat L.E. Nat. Struct. Mol. Biol. 2004; 11: 992-1000Crossref PubMed Scopus (79) Google Scholar) and references therein). Likewise, PABPN1 is replaced upon transport of the mRNA to the cytoplasm by the cytoplasmic poly(A)-binding protein (PABPC, Pab1p in Saccharomyces cerevisiae). PABPC is one of the key factors that participates in mediating several of the roles the mRNA poly(A) tail has on gene expression, including its stimulatory effect on translation (see Refs. 4.Sachs A.B. Varani G. Nat. Struct. Biol. 2000; 7: 356-361Crossref PubMed Scopus (104) Google Scholar and 8.Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (257) Google Scholar and references therein). Both the cap and the poly(A) tail play critical roles during eukaryotic mRNA degradation, and in two of the general pathways poly(A), removal precedes the degradation of the cap (2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar, 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar, 5.Wilusz C.J. Wormington M. Peltz S.W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (628) Google Scholar). The chemical nature of the cap makes the mRNA 5′ end inaccessible to 5′-exoribonucleases, and specific decapping activities are required for its degradation (reviewed in Ref. 13.Coller J. Parker R. Annu. Rev. Biochem. 2004; 73: 861-890Crossref PubMed Scopus (394) Google Scholar). In the deadenylation-dependent decapping pathway, the cap is removed by the Dcp1p/Dcp2p decapping activity after the initial deadenylation step, whereas the scavenger decapping enzyme DcpS degrades the cap as one of the final steps in the deadenylation followed by the 3′-5′ degradation pathway. Several different poly(A)-degrading activities (reviewed in Refs. 2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar and 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar) have been identified in eukaryotic cells, although their roles in the different mRNA degradation pathways have not yet been worked out. At least three of these activities are highly poly(A)-specific: the Pan2/3 nuclease in yeast and mammals (14.Boeck 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 (146) Google Scholar, 15.Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), poly(A)-specific ribonuclease (PARN) in vertebrate cells (16.Åström J. Åström A. Virtanen A. EMBO J. 1991; 10: 3067-3071Crossref PubMed Scopus (75) Google Scholar, 17.Copeland P.R. Wormington M. RNA (N. Y.). 2001; 7: 875-886Crossref PubMed Scopus (89) Google Scholar, 18.Körner C.G. Wahle E. J. Biol. Chem. 1997; 272: 10448-10456Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19.Körner C.G. Wormington M. Muckenthaler M. Schneider S. Dehlin E. Wahle E. EMBO J. 1998; 17: 5427-5437Crossref PubMed Scopus (203) Google Scholar, 20.Martinez J. Ren Y.G. Thuresson A.C. Hellman U. Astrom J. Virtanen A. J. Biol. Chem. 2000; 275: 24222-24230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and Arabidopsis (21.Chiba Y. Johnson M.A. Lidder P. Vogel J.T. van Erp H. Green P.J. Gene (Amst.). 2004; 328: 95-102Crossref PubMed Scopus (57) Google Scholar), and the Ccr4-Pop2-Not complex in yeast (22.Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Crossref PubMed Scopus (165) Google Scholar, 23.Tucker 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 (467) Google Scholar) and its orthologs in other organisms (see Refs. 2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar and 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar and references therein). Mechanisms controlling mRNA degradation are still poorly understood at the molecular level, although recent studies have begun to unravel the components involved in modulating RNA degradation (reviewed in Refs. 2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar, 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar, and 5.Wilusz C.J. Wormington M. Peltz S.W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (628) Google Scholar). Among the cis-acting elements, the AU-rich elements (ARE) (24.Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3121) Google Scholar) (reviewed in Refs. 5.Wilusz C.J. Wormington M. Peltz S.W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (628) Google Scholar and 25.Chen C.-Y.A. Shyu A.-B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1677) Google Scholar), which are frequently located in the 3′-untranslated regions of mRNA, are known to interact with a variety of RNA-binding proteins and participate in regulating mRNA turnover. Besides the AREs, the poly(A) tail is also a primary target at which regulation of mRNA decay occurs, and PABPC/Pab1p has turned out to be both a positive and a negative regulator of poly(A) degradation (2.Meyer S. Temme C. Wahle E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 197-216Crossref PubMed Scopus (288) Google Scholar, 3.Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (645) Google Scholar). The Pan2/3 nuclease activity is positively affected by PABPC/Pab1p binding to the poly(A) (15.Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 26.Sachs A.B. Deardorff J.A. Cell. 1992; 70: 961-973Abstract Full Text PDF PubMed Scopus (159) Google Scholar) tail, whereas the activities of PARN (18.Körner C.G. Wahle E. J. Biol. Chem. 1997; 272: 10448-10456Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and yeast Ccr4-Pop2-Not complex (28.Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (266) Google Scholar, 29.Viswanathan P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; 278: 14949-14955Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) are inhibited by PABPC/Pab1p. In addition to this, it is also known that the poly(A) tail affects decapping (30.Caponigro G. Parker R. Genes Dev. 1995; 9: 2421-2432Crossref PubMed Scopus (230) Google Scholar, 31.Coller J.M. Gray N.K. Wickens M.P. Genes Dev. 1998; 12: 3226-3235Crossref PubMed Scopus (170) Google Scholar, 32.Wilusz C.J. Gao M. Jones C.L. Wilusz J. Peltz S.W. RNA (N. Y.). 2001; 7: 1416-1424PubMed Google Scholar). In the human case, experimental evidence suggests that PABPC protects the cap from Dcp2-mediated decapping by binding simultaneously to both the cap structure and the poly(A) tail (33.Khanna R. Kiledjian M. EMBO J. 2004; 23: 1968-1976Crossref PubMed Scopus (52) Google Scholar). Here we have investigated how the nuclear and cytoplasmic cap-binding proteins CBC and eIF4E affect human PARN-mediated degradation of the poly(A) tail at the 3′ end of the mRNA. PARN is unique among the major poly(A) nucleases since it interacts directly with both the poly(A) tail at the 3′ end of the mRNA and the 5′ end-located cap structure during poly(A) hydrolysis (20.Martinez J. Ren Y.G. Thuresson A.C. Hellman U. Astrom J. Virtanen A. J. Biol. Chem. 2000; 275: 24222-24230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 34.Dehlin E. Wormington M. Körner C.G. Wahle E. EMBO J. 2000; 19: 1079-1086Crossref PubMed Scopus (156) Google Scholar, 35.Martinez J. Ren Y.G. Nilsson P. Ehrenberg M. Virtanen A. J. Biol. Chem. 2001; 276: 27923-27929Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We found that CBC inhibited PARN activity. Our data, therefore, suggested a previously unrecognized function for CBC in mRNA deadenylation, identified CBC as a potential regulator of PARN, and once again emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Expression and Purification of Recombinant PARN, CBC, and eIF4E—Recombinant and His-tagged versions of human PARN, PARN(W475A), PARN(E455A,W456A), and PARN(1–470) were expressed and purified to apparent homogeneity as described previously (20.Martinez J. Ren Y.G. Thuresson A.C. Hellman U. Astrom J. Virtanen A. J. Biol. Chem. 2000; 275: 24222-24230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Recombinant human CBP20 and CBP80 were purified, and CBC was reconstituted according to Mazza et al. (36.Mazza C. Ohno M. Segref A. Mattaj I.W. Cusack S. Mol. Cell. 2001; 8: 383-396Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 37.Mazza C. Segref A. Mattaj I.W. Cusack S. EMBO J. 2002; 21: 5548-5557Crossref PubMed Scopus (160) Google Scholar). His-tagged eIF4E was expressed in Escherichia coli strain BL21 (DE3) containing plasmid pET15-eIF4E 5J. Martinez and A. Virtanen, unpublished results. and purified to apparent homogeneity using the same protocol as was used for purification of His-tagged PARN (20.Martinez J. Ren Y.G. Thuresson A.C. Hellman U. Astrom J. Virtanen A. J. Biol. Chem. 2000; 275: 24222-24230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In plasmid pET15-eIF4E, the open reading frame of the human eIF4E mRNA was inserted between the NdeI and BamHI restriction enzyme sites of the vector pET-15b (Novagen Inc.). The reading frame of eIF4E was in-frame with an N-terminally located His tag originating from the cloning vector. Preparation of RNA Substrates—RNA substrates (16.Åström J. Åström A. Virtanen A. EMBO J. 1991; 10: 3067-3071Crossref PubMed Scopus (75) Google Scholar) with or without m7G(5′)ppp(5′)G at their 5′ ends were synthesized by in vitro transcription according to Martinez et al. (35.Martinez J. Ren Y.G. Nilsson P. Ehrenberg M. Virtanen A. J. Biol. Chem. 2001; 276: 27923-27929Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). For body labeling, the specific radioactivities of the included radioactive mononucleotides were 40 Ci/mmol, whereas for poly(A) tail labeling, the specific radioactivity was 5 Ci/mmol. Deadenylation Assays—The indicated amounts of capped (m7GpppG) or non-capped 32P-labeled L3(A30) RNA were incubated under standard in vitro deadenylation conditions (20 mm HEPES-KOH (pH 7), 1.5 mm MgCl2, 100 mm KCl, 0.1 mm EDTA, 0.5 mm dithiothreitol, 2.5% polyvinyl alcohol, 10% glycerol, and 0.1–0.2 units of RNAguard™ RNase inhibitor (Amersham Biosciences, catalog number 27-0815-01)) as described previously (35.Martinez J. Ren Y.G. Nilsson P. Ehrenberg M. Virtanen A. J. Biol. Chem. 2001; 276: 27923-27929Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Reactions were mixed at 4 °C and contained the indicated amounts of PARN, eIF4E, CBP20, CBP80, and/or reconstituted CBC. The final concentrations of CBC and eIF4E in the reactions were chosen to be in the range of their dissociation constant values, being ∼4 and 100 nm, respectively (38.Niedzwiecka A. Marcotrigiano J. Stepinski J. Jankowska-Anyszka M. Wyslouch-Cieszynska A. Dadlez M. Gingras A.C. Mak P. Darzynkiewicz E. Sonenberg N. Burley S.K. Stolarski R. J. Mol. Biol. 2002; 319: 615-635Crossref PubMed Scopus (317) Google Scholar, 39.Worch R. Niedzwiecka A. Stepinski J. Mazza C. Jankowska-Anyszka M. Darzynkiewicz E. Cusack S. Stolarski R. RNA (N. Y.). 2005; 11: 1355-1363Crossref PubMed Scopus (55) Google Scholar). The reaction volume was 21 μl, and incubations were performed at 30 °C for 30 min. The reaction products were analyzed by TLC or purified and analyzed on 10% polyacrylamide (acrylamide/bisacrylamide 19:1, v/v)/7 m urea gels as described elsewhere (16.Åström J. Åström A. Virtanen A. EMBO J. 1991; 10: 3067-3071Crossref PubMed Scopus (75) Google Scholar). One-dimensional TLC Assays—Deadenylation reactions with increasing amounts of CBC or eIF4E, as indicated, were performed as described above. Capped (m7GpppG) or non-capped 32P-poly(A) tail-labeled L3(A30) RNA was used, as indicated. The released 5′-AMP product was detected by TLC analysis. Essentially, a 1-μl aliquot taken from the 21 μl-reaction was spotted onto a polyethyleneimine cellulose F plastic sheet (Merck, catalog number 1.05579), and the chromatogram was developed using 0.75 m KH2PO4, pH 3.5 (H3PO4), as solvent. The plate was dried and scanned by a 400S PhosphorImager (Amersham Biosciences). For the kinetic analysis, 0.1 mg/ml bovine serum albumin was added in the reactions, and analysis was performed as described elsewhere (35.Martinez J. Ren Y.G. Nilsson P. Ehrenberg M. Virtanen A. J. Biol. Chem. 2001; 276: 27923-27929Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Immunoprecipitation—Co-immunoprecipitation assays were performed according to McKendrick et al. (40.McKendrick L. Thompson E. Ferreira J. Morley S.J. Lewis J.D. Mol. Cell. Biol. 2001; 21: 3632-3641Crossref PubMed Scopus (98) Google Scholar). HeLa nuclear extracts were purchased from 4C (Mons, Belgium) and subjected to immunoprecipitation using affinity-purified anti-PARN or anti-human CBP80(116) (7.Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar) polyclonal serum. The polyclonal antiserum specific for human PARN was generated by immunizing rabbits using 0.45 mg/rabbit recombinant purified His-tagged PARN polypeptide followed by three boost injections with the same amount of antigen. The extracts were treated before use with 50 μl of protein A-Sepharose CL-4B beads (Amersham Biosciences) in 4 °C for 1 h with rotation followed by centrifugation at 10,000 × g in 4 °C for 10 min. The cleared supernatant was diluted 5-fold in IP150 buffer (20 mm HEPES, pH 7.9, 150 mm KCl, 0.05% Nonidet P-40, and 1% Triton X-100) and incubated with an appropriate volume of the antibody/antiserum in 4 °C for 1 h with rotation. Subsequently, the protein A-Sepharose beads were recovered by centrifugation and incubated at 4 °C with 50 μg of RNase A/ml for 10 min. Finally, the beads were washed three times with 1 ml of ice-cold IP150 buffer and eluted with SDS-PAGE sample buffer, and the recovered proteins were resolved by SDS-PAGE. The resulting gel was transferred to polyvinylidene difluoride membrane, following a standard Western blot procedure (41.Jareborg N. Burnett S. J. Gen. Virol. 1991; 72: 2269-2274Crossref PubMed Scopus (16) Google Scholar), and probed with the indicated antibody. CBC Inhibits PARN Activity—To investigate whether the nuclear (i.e. CBC) or cytoplasmic (i.e. eIF4E) cap-binding proteins had any effect on human PARN activity, we incubated a polyadenylated and 7-methyl guanosine capped RNA substrate, L3(A30), in the presence of PARN and a 1:1 in vitro reconstituted CBC complex or eIF4E. We found that CBC inhibited PARN activity and that the inhibition effect was titratable, whereas eIF4E had no detectable effect on PARN (Fig. 1, A and B, see also below and see Fig. 4). It has previously been observed by Wilusz and co-workers (27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) that eIF4E inhibits PARN-mediated deadenylation in an in vitro decay system based on a crude HeLa cell-free S100 extract. Thus, as compared with the study by Wilusz and co-workers (27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), we were not able to reconstitute the eIF4E inhibition effect on PARN in our purified system, even if we increased the eIF4E concentration far above the concentrations used in their study. This discrepancy is most likely explained by the use of different in vitro deadenylation systems. We have used a highly purified and defined reconstituted deadenylation system based on recombinant components, whereas Wilusz and co-workers (27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) used a crude in vitro decay system. It is therefore likely that the eIF4E inhibition effect they observed requires additional components that are not present in our highly purified in vitro system.FIGURE 4Kinetic analysis of mechanism of inhibition. A, 0.5–60 nm m7GpppG-capped L3(A30) RNA, 32P-labeled in its poly(A) tail, was incubated for 30 min with 0.6 nm PARN in the absence of (open circles) or in the presence of 5 (open squares), 10 (open triangles), or 20 nm (crosses) of reconstituted CBC. The release of adenosine monophosphates was monitored by one-dimensional TLC assay, as described under “Materials and Methods.” The reciprocal initial velocity (1/v) is plotted versus reciprocal RNA concentration (1/S). B, same as A, with the difference that non-capped L3(A30) RNA 32P-labeled in its poly(A) tail was used instead. CBC was absent (filled circles) or present at 5 (filled squares), 10 (filled triangles), or 20 nm (filled circles with x). C, same as A, except that 0.6 nm PARN(W475A) was used as the deadenylating activity in the absence of (open circles) or in the presence of 5 (open squares), 10 (open triangles), or 20 nm (crosses) of reconstituted CBC. D, same as C with the difference that non-capped L3(A30) RNA 32P-labeled in its poly(A) tail was used instead. CBC was absent (filled circles) or present at 5 (filled squares), 10 (filled triangles), or 20 nm (filled circles with x). E, same as A, except that the reactions were performed in the absence of (open circles) or in the presence of 5 (open squares), 10 (open triangles), or 20 nm (crosses) of CBP80. F, same as E, with the difference that non-capped L3(A30) RNA 32P-labeled in its poly(A) tail was used instead. CBP80 was absent (filled circles) or present at 5 (filled squares), 10 nm (filled triangles), or 20 nm (filled circles with x).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further examine the CBC inhibition effect, we investigated whether it was cap-dependent and found that CBC inhibited PARN activity even if the RNA substrate lacked a cap structure (Fig. 1C). This suggests that CBC may inhibit PARN through a direct interaction without binding the cap. To investigate whether this could be the case, we incubated PARN with CBC at an RNA substrate concentration that was at least five times higher than the Km value for recombinant PARN (42.Ren Y.G. Martinez J. Kirsebom L.A. Virtanen A. RNA (N. Y.). 2002; 8: 1393-1400Crossref PubMed Scopus (31) Google Scholar), using both capped and non-capped substrates. Fig. 1, B and C, shows that CBC could still inhibit PARN under these conditions, providing further support that CBC inhibits PARN through a direct and cap-independent interaction. It has previously been shown that PARN interacts with the 5′ end cap structure (20.Martinez J. Ren Y.G. Thuresson A.C. Hellman U. Astrom J. Virtanen A. J. Biol. Chem. 2000; 275: 24222-24230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.Gao M. Fritz D.T. Ford L.P. Wilusz J. Mol. Cell. 2000; 5: 479-488Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 34.Dehlin E. Wormington M. Körner C.G. Wahle E. EMBO J. 2000; 19: 1079-1086Crossref PubMed Scopus (156) Google Scholar, 35.Martinez J. Ren Y.G. Nilsson P. Ehrenberg M. Virtanen A. J. Biol. Chem. 2001; 276: 27923-27929Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and that this interaction stimulates PARN activity by increasing the rate of hydrolysis, which in turn amplifies PARN processivity. It was therefore of interest to investigate whether the cap binding property of PARN influenced CBC inhibition of PARN activity. For this purpose, we used three PARN mutants, the point mutant PARN(W475A), the double point mutant PARN(E455A,W456A), and the C-terminally deletion mutant PARN(1–470), all being active in deadenylation (Fig. 2) but severely defective in their cap binding properties (supplemental Table 1). Fig. 2 shows that mutants PARN(W475A) and PARN(E455A,W456A) were efficiently inhibited by CBC, whereas the deletion mutant PARN (1–470) was unaffected. From these data, we conclude that the C-terminal region of PARN is essential, whereas the cap binding property of PARN is not a prerequisite for CBC-mediated inhibition. Importantly, the PARN(1–470) data show that PARN-mediated deadenylation can occur in the presence of CBC. Thus, the CBC inhibitory effect was not caused by CBC interfering with the RNA substrate, for example, by making the RNA substrate inaccessible to PARN hydrolysis (see also below and see Fig. 4). The 80-kDa Subunit of CBC Inhibits PARN Activity—CBC is composed of two subunits, CBP20 and CBP80 (7.Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (430) Google Scholar). The primary function of CBP20 (36.Mazza C. Ohno M. Segref A. Mattaj I.W. Cusack S. Mol. Cell. 2001; 8: 383-396Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 43.Calero G. Wilson K.F. Ly T. Rios-Steiner J.L. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 2002; 9: 912-917Crossref PubMed Scopus (124) Google Scholar) is to physically interact with the cap, whereas CBP80 has at least two separate functions (37.Mazza C. Segref A. Mattaj I.W. Cusack S. EMBO J. 2002; 21: 5548-5557Crossref PubMed Scopus (160) Google Scholar): (i) to interact with and induce a conformational change of CBP20 that facilitates CBP20/cap interaction and (ii) to provide a large surface for binding to proteins involved in different aspects of mRNA and cap-dependent functions, such as interaction with translation factor eIF4G (12.Lejeune F. Ranganathan A.C. Maquat L.E. Nat. Struct. Mol. Biol. 2004; 11: 992-1000Crossref PubMed Scopus (79) Google Scholar, 40.McKendrick L. Thompson E. Ferreira J. Morley S.J. Lewis J.D. Mol. Cell. Biol. 2001; 21: 3632-3641Crossref PubMed Scopus (98) Google Scholar, 44.Fortes P. Inada T. Preiss T. Hentze M.W. Mattaj I.W. Sachs A.B. Mol. Cell. 2000; 6: 191-196Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) or phosphorylated adaptor for RNA export (45.Ohno M. Segref A. Bachi A. Wilm M. Mattaj I.W. Cell. 2000; 101: 187-198Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In light of this, we investigated whether the entire CBC complex was required for PARN inhibition or whether either of the two subunits alone could inhibit PARN. Fig. 3A shows that CBP80 inhibited PARN in a cap-independent manner, whereas CBP20 had no effect on PARN activity. As expected, we also found that both cap binding site mutants, PARN(E455A,W456A) and PARN(W475A), were inhibited by CBP80, whereas the C-terminal deletion mutant PARN(1–470) was unaffected by the addition of CBP80 (Fig. 3B). These data, which are in keeping with the observation that CBC inhibition of PARN is cap-independent, show that CBP80 alone inhibits PARN activity and strongly suggest that the inhibition" @default.
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• W2071720275 date "2006-02-01" @default.
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• W2071720275 title "Inhibition of mRNA Deadenylation by the Nuclear Cap Binding Complex (CBC)" @default.
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