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• W2039659797 abstract "The CCA-adding enzyme builds and repairs the 3′ terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3′-terminal CCA, as do all mature tRNAs; the other 35% ends in 3′ CC or possibly 3′ C. The 3′-terminal A of U2 snRNA cannot be encoded because the 3′ end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10–16 extra 3′ nucleotides that are removed by one or more 3′ exonucleases. Thus, if 3′ exonuclease activity removes the encoded 3′ CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3′-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3′-terminal A, CA, or CCA to human U2 snRNA lacking 3′-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with theEscherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3′ stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun. The CCA-adding enzyme builds and repairs the 3′ terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3′-terminal CCA, as do all mature tRNAs; the other 35% ends in 3′ CC or possibly 3′ C. The 3′-terminal A of U2 snRNA cannot be encoded because the 3′ end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10–16 extra 3′ nucleotides that are removed by one or more 3′ exonucleases. Thus, if 3′ exonuclease activity removes the encoded 3′ CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3′-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3′-terminal A, CA, or CCA to human U2 snRNA lacking 3′-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with theEscherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3′ stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun. small nuclear RNA nucleotide(s) signal recognition particle The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) synthesizes and regenerates the 3′-terminal CCA sequence of tRNA by adding three consecutive nucleotides in the order of C, C, and A (1Deutscher M.P. Methods Enzymol. 1990; 181: 434-439Crossref PubMed Scopus (15) Google Scholar, 2Sprinzl M. Cramer F. Prog. Nucleic Acid Res. Mol. Biol. 1979; 22: 1-69Crossref PubMed Scopus (133) Google Scholar, 3Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908PubMed Google Scholar). The CCA-adding enzyme is the only polymerase known that can synthesize a defined sequence without using a nucleic acid template (4Deutscher M.P. Methods Enzymol. 1974; 29: 706-716Crossref PubMed Scopus (11) Google Scholar,5Shi P.Y. Maizels N. Weiner A.M. EMBO J. 1998; 17: 3197-3206Crossref PubMed Scopus (98) Google Scholar). The enzyme is essential in eukaryotes, archaea, and some eubacteria, where many or all tRNA genes lack encoded CCA (6Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2279) Google Scholar, 7Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Reeve J.N. et al.J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1036) Google Scholar). In these organisms, 3′ trailer sequences are removed from tRNA precursors by nucleases that stop at the discriminator base (position 73), leaving the acceptor stem intact as a substrate for the CCA-adding enzyme (3Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908PubMed Google Scholar,6Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2279) Google Scholar, 7Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Reeve J.N. et al.J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1036) Google Scholar). In Escherichia coli and presumably in other eubacteria in which all tRNA genes encode CCA, the CCA-adding enzyme is not essential but is advantageous (8Zhu L. Deutscher M.P. EMBO J. 1987; 6: 2473-2477Crossref PubMed Scopus (88) Google Scholar) because it can repair CCA termini depleted by exonuclease activity (9Li Z. Deutscher M.P. J. Biol. Chem. 1994; 269: 6064-6071Abstract Full Text PDF PubMed Google Scholar, 10Li Z. Pandit S. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2856-2861Crossref PubMed Scopus (162) Google Scholar). The ability of CCA-adding enzymes to recognize all cytoplasmic tRNAs regardless of amino acid acceptor specificity (11Giege R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12078-12081Crossref PubMed Scopus (36) Google Scholar) and the ability of synthetic tDNAs to serve as substrates for the E. coli CCA-adding enzyme (5Shi P.Y. Maizels N. Weiner A.M. EMBO J. 1998; 17: 3197-3206Crossref PubMed Scopus (98) Google Scholar, 12Shi P.Y. Weiner A.M. Maizels N. RNA (N. Y.). 1998; 4: 276-284PubMed Google Scholar) suggest that recognition involves structural features common to all but some unusual organellar tRNAs (13Nagaike T. Suzuki T. Tomari Y. Takemoto-Hori C. Negayama F. Watanabe K. Ueda T. J. Biol. Chem. 2001; 276: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).U2 small nuclear RNA (snRNA)1is a small, highly conserved, nonpolyadenylated nuclear RNA that plays an essential role in mRNA splicing (14Gesteland R.F. Atkins J.F. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World (Monograph 24). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993Google Scholar, 15Gesteland R.F. Cech T. Atkins J.F. The RNA World (Monograph 37).2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar). In vertebrates, the genes for all snRNAs of the Sm class (snRNAs that bind Sm antigens) and some small nucleolar RNAs of the box C+D class such as U3 (16Weinstein L.B. Steitz J.A. Curr. Opin. Cell Biol. 1999; 11: 378-384Crossref PubMed Scopus (249) Google Scholar) share a common transcriptional apparatus: transcription is driven by a specialized RNA polymerase II promoter, consisting of an enhancer-like distal sequence element and a TATA-like proximal sequence element that are spaced almost precisely one nucleosome apart (17Ma B. Hernandez N. J. Biol. Chem. 2001; 276: 5027-5035Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and appear to be brought together by a bound nucleosome (18Boyd D.C. Greger I.H. Murphy S. Gene (Amst.). 2000; 247: 33-44Crossref PubMed Scopus (26) Google Scholar, 19Zhao X. Pendergrast P.S. Hernandez N. Mol. Cell. 2001; 7: 539-549Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). A highly conserved 3′ end formation signal or “3′ box” (20Ach R.A. Weiner A.M. Mol. Cell. Biol. 1987; 7: 2070-2079Crossref PubMed Scopus (33) Google Scholar, 21Hernandez N. EMBO J. 1985; 4: 1827-1837Crossref PubMed Scopus (108) Google Scholar, 22Yuo C.Y. Ares Jr., M. Weiner A.M. Cell. 1985; 42: 193-202Abstract Full Text PDF PubMed Scopus (65) Google Scholar) located some 6–25 nucleotides beyond the snRNA coding region directs formation of the first detectable U snRNA precursors (U2+10) containing 10–16 extra nucleotides and ending just upstream of the 3′ box. However, the 3′ box functions only when transcription is driven by a specialized U snRNA promoter; mRNA promoters cannot substitute for the U snRNA promoter (17Ma B. Hernandez N. J. Biol. Chem. 2001; 276: 5027-5035Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 23de Vegvar H.E. Lund E. Dahlberg J.E. Cell. 1986; 47: 259-266Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 24Hernandez N. Weiner A.M. Cell. 1986; 47: 249-258Abstract Full Text PDF PubMed Scopus (160) Google Scholar).Although U2 (but not U1) transcription appears to continue past the 3′ box (25Cuello P. Boyd D.C. Dye M.J. Proudfoot N.J. Murphy S. EMBO J. 1999; 18: 2867-2877Crossref PubMed Scopus (50) Google Scholar), it remains to be seen whether the 3′ box functions as a transcription termination signal, as observed for rRNA transcription by RNA polymerase I (26Bartsch I. Schoneberg C. Grummt I. Mol. Cell. Biol. 1987; 7: 2521-2529Crossref PubMed Scopus (27) Google Scholar), or as an RNA processing signal, like the AAUAAA element of polyadenylated mRNAs (27Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (180) Google Scholar, 28Keller W. Cell. 1995; 81: 829-832Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 29Wahle E. Keller W. Annu. Rev. Biochem. 1992; 61: 419-440Crossref PubMed Google Scholar) and the CAGA box of nonpolyadenylated replicative histone mRNAs (30Dominski Z. Erkmann J.A. Greenland J.A. Marzluff W.F. Mol. Cell. Biol. 2001; 21: 2008-2017Crossref PubMed Scopus (38) Google Scholar). U2+10 is subsequently exported to the cytoplasm, where the 3′ tail is trimmed (31Booth Jr., B.L. Pugh B.F. J. Biol. Chem. 1997; 272: 984-991Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 32Eliceiri G.L. Sayavedra M.S. Biochem. Biophys. Res. Commun. 1976; 72: 507-512Crossref PubMed Scopus (38) Google Scholar, 33Huang Q. Jacobson M.R. Pederson T. Mol. Cell. Biol. 1997; 17: 7178-7185Crossref PubMed Scopus (19) Google Scholar, 34Kleinschmidt A.M. Pederson T. Mol. Cell. Biol. 1987; 7: 3131-3137Crossref PubMed Scopus (23) Google Scholar) by endonuclease and exonuclease activities that could be related to the yeast exosome, a multinuclease complex involved in processing many cellular RNAs (35Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (488) Google Scholar, 36Allmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (374) Google Scholar). Sm antigens then assemble onto the Sm binding site, and the resulting immature U2 snRNP is imported into the nucleus for final assembly into a mature U2 snRNP (35Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (488) Google Scholar,37Friesen W.J. Massenet S. Paushkin S. Wyce A. Dreyfuss G. Mol. Cell. 2001; 7: 1111-1117Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 38Huang Q. Pederson T. Nucleic Acids Res. 1999; 27: 1025-1031Crossref PubMed Scopus (23) Google Scholar, 39Will C.L. Luhrmann R. Curr. Opin. Cell Biol. 2001; 13: 290-301Crossref PubMed Scopus (548) Google Scholar). Addition of U2 snRNP-specific antigens may occur in Cajal bodies (also known as coiled bodies or CBs) (40Matera A.G. Trends Cell Biol. 1999; 9: 302-309Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 41Narayanan A. Speckmann W. Terns R. Terns M.P. Mol. Biol. Cell. 1999; 10: 2131-2147Crossref PubMed Scopus (114) Google Scholar, 42Sleeman J.E. Lamond A.I. Curr. Biol. 1999; 9: 1065-1074Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), whereas nucleotide modifications (methylation and pseudouridylation) occur in the nucleolus (43Yu Y.T. Shu M.D. Narayanan A. Terns R.M. Terns M.P. Steitz J.A. J. Cell Biol. 2001; 152: 1279-1288Crossref PubMed Scopus (52) Google Scholar).Approximately 65% of mature human U2 snRNA ends in 3′-terminal CCA, as do all mature tRNAs; the other 35% ends in 3′ CC or possibly 3′ C, as these are not easily distinguished experimentally (44Perumal K. Gu J. Reddy R. Mol. Cell. Biochem. 2000; 208: 99-109Crossref PubMed Google Scholar, 45Sinha K.M. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The 3′-terminal A of U2 snRNA must be added posttranscriptionally because the U2 snRNA coding region ends with CC/CC, where the slashindicates the last encoded nucleotide (45Sinha K.M. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 46Chen Y. Sinha K. Perumal K. Reddy R. RNA (N. Y.). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar, 47Pavelitz T. Rusche L. Matera A.G. Scharf J.M. Weiner A.M. EMBO J. 1995; 14: 169-177Crossref PubMed Scopus (63) Google Scholar). If 3′ exonucleolytic processing removes the encoded 3′ CC during U2 snRNA maturation, as appears to be the case in vitro (33Huang Q. Jacobson M.R. Pederson T. Mol. Cell. Biol. 1997; 17: 7178-7185Crossref PubMed Scopus (19) Google Scholar, 34Kleinschmidt A.M. Pederson T. Mol. Cell. Biol. 1987; 7: 3131-3137Crossref PubMed Scopus (23) Google Scholar, 38Huang Q. Pederson T. Nucleic Acids Res. 1999; 27: 1025-1031Crossref PubMed Scopus (23) Google Scholar), then the cell may need to build or rebuild the 3′-terminal A, CA, or CCA of U2 snRNA. Using a variety of synthetic and natural U2 snRNAs as substrate, we show here that the human but not the E. coli CCA-adding enzyme can build and repair the 3′-terminal CCA sequence of human U2 snRNA. The human CCA-adding enzyme is also active on native U2 snRNPs. The yeast (48Wolfe C.L. Hopper A.K. Martin N.C. J. Biol. Chem. 1996; 271: 4679-4686Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and vertebrate CCA-adding enzymes (49Hopper A.K. Science. 1998; 282: 2003-2004Crossref PubMed Scopus (17) Google Scholar, 50Lund E. Dahlberg J.E. Science. 1998; 282: 2082-2085Crossref PubMed Scopus (267) Google Scholar) are known to be present in the nucleus as well as the cytoplasm, consistent with the possibility that the human CCA-adding enzyme may build the 3′ end of immature U2 snRNPs in the cytoplasm or nucleus or repair the 3′ end of mature nuclear U2 snRNPs.DISCUSSIONAll vertebrate U snRNAs belonging to the Sm class of U snRNPs and some small nucleolar RNAs such as U3 small nucleolar RNA apparently use the same 3′ end formation apparatus: specialized U snRNA promoter elements drive transcription by a form of RNA polymerase II that allows recognition of a highly conserved 3′ end formation signal (3′ box) located 6–25 nt downstream from the U snRNA coding (20Ach R.A. Weiner A.M. Mol. Cell. Biol. 1987; 7: 2070-2079Crossref PubMed Scopus (33) Google Scholar). Whether the 3′ box functions as a transcription termination signal or as an RNA processing signal is still unknown (25Cuello P. Boyd D.C. Dye M.J. Proudfoot N.J. Murphy S. EMBO J. 1999; 18: 2867-2877Crossref PubMed Scopus (50) Google Scholar); however, the first detectable U snRNA precursors have 3′-terminal tails that extend nearly to the 3′ box (22Yuo C.Y. Ares Jr., M. Weiner A.M. Cell. 1985; 42: 193-202Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 31Booth Jr., B.L. Pugh B.F. J. Biol. Chem. 1997; 272: 984-991Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 32Eliceiri G.L. Sayavedra M.S. Biochem. Biophys. Res. Commun. 1976; 72: 507-512Crossref PubMed Scopus (38) Google Scholar, 35Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (488) Google Scholar, 36Allmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (374) Google Scholar, 71Stroke I.L. Weiner A.M. J. Mol. Biol. 1985; 184: 183-193Crossref PubMed Scopus (43) Google Scholar) and are trimmed by cytoplasmic nucleases that may be related to the yeast multinuclease complex known as the exosome (10Li Z. Pandit S. Deutscher M.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2856-2861Crossref PubMed Scopus (162) Google Scholar, 35Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (488) Google Scholar, 36Allmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (374) Google Scholar, 38Huang Q. Pederson T. Nucleic Acids Res. 1999; 27: 1025-1031Crossref PubMed Scopus (23) Google Scholar).The 3′-terminal sequence of most U snRNAs can be aligned with the corresponding genes, suggesting that nucleases are capable of generating the mature 3′ end of most U snRNA precursors. However, ∼65% of mature human U2 snRNA ends in 3′-terminal CCA; the other 35% ends in 3′ CC or possibly 3′ C (44Perumal K. Gu J. Reddy R. Mol. Cell. Biochem. 2000; 208: 99-109Crossref PubMed Google Scholar, 45Sinha K.M. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The 3′-terminal A of U2 snRNA cannot be encoded because the 3′ end of the U2 snRNA gene is CC/CC, where the slash indicates the last encoded nucleotide (47Pavelitz T. Rusche L. Matera A.G. Scharf J.M. Weiner A.M. EMBO J. 1995; 14: 169-177Crossref PubMed Scopus (63) Google Scholar). The 3′-terminal A is also unlikely to be an untemplated addition by the transcribing RNA polymerase (72Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1878) Google Scholar) because transcription must continue for >10 bp beyond the 3′ end to generate the U2+10 family of precursors. We cannot exclude template-instructed misincorporation, as occurs in certain RNA viruses (73Cattaneo R. Annu. Rev. Genet. 1991; 25: 71-88Crossref PubMed Scopus (90) Google Scholar), but this has never been observed for a cellular RNA polymerase. Thus, the solitary 3′-terminal A of U2 snRNA is almost surely added posttranscriptionally.One candidate for posttranscriptional addition of 3′-terminal A to U2 snRNA is the SRP 7SL adenylating enzyme (62Perumal K. Sinha K. Henning D. Reddy R. J. Biol. Chem. 2001; 276: 21791-21796Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); another is the CCA-adding enzyme (tRNA nucleotidyltransferase), which builds and repairs the 3′-terminal CCA of tRNA. In fact, the 3′ end of U2 strongly resembles the minimal substrate for the CCA-adding enzyme: a tRNA minihelix (the “top half ”of tRNA) in which the acceptor stem stacks on the TψC stem/loop. Not only is U2 stem IV 12 bp, the optimal length for CCA addition by the E. coli and S. shibatae enzymes to a minihelix substrate (12Shi P.Y. Weiner A.M. Maizels N. RNA (N. Y.). 1998; 4: 276-284PubMed Google Scholar), but the stem is followed by A185CCAOH, where A185 is formally analogous to the discriminator base of tRNA, most frequently a purine (74Crothers D.M. Seno T. Soll G. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3063-3067Crossref PubMed Scopus (182) Google Scholar). Thus, in principle, the CCA-adding enzyme might be capable of completely rebuilding the 3′-terminal CCA sequence of U2 snRNA if nucleases overprocessed the U2 snRNA precursor or other 3′ exonucleases attacked the mature U2 snRNA.To ask whether CCA-adding enzymes can add 3′-terminal A, CA, or CCA to U2 snRNA precursors, we used three sets of substrates (full-length U2 snRNA, 3′-terminal stem/loops III and IV, and stem/loop IV alone), each lacking 3′-terminal A, CA, or CCA. We assayed these substrates with CCA-adding enzymes of both classes from all three kingdoms (3Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908PubMed Google Scholar): class I CCA-adding enzymes from archaea (S. shibatae, M. jannaschii, P. furiosus, and M. thermoautotrophicum), class II enzymes from Gram-positive (B. stearothermophilus and B. subtilis) and Gram-negative (E. coli) eubacteria, and the human class II enzyme. All substrates were active with the human class II CCA-adding enzyme, including 3′-terminal stem/loop IV alone (Figs. Figure 1, Figure 2, Figure 3), and CCA addition was faithful (Fig. 2E); however, the E. coli class II CCA-adding enzyme was inactive on the U2 snRNA substrates but fully active on tRNA substrates, consistent with the idea that CCA addition to U2 snRNA is specific. In contrast to CCA-adding enzymes, the SRP 7SL RNA adenylating enzyme nonspecifically added long 3′-terminal tails of >400 nt to U2 and other RNA substrates (Fig. 4). Thus, the SRP RNA adenylating enzyme behaves like a poly(A) polymerase and is unlikely to play a role in U2 snRNA biosynthesis. These results suggest that the 3′ stem/loop of U2 snRNA does in fact resemble a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes (12Shi P.Y. Weiner A.M. Maizels N. RNA (N. Y.). 1998; 4: 276-284PubMed Google Scholar, 57Hou Y.M. RNA (N. Y.). 2000; 6: 1031-1043Crossref PubMed Scopus (30) Google Scholar), and that CCA addition to U2 snRNA could take place in vivo.We also found that the human CCA-adding enzyme is active on natural U2 snRNA purified from nuclear extract (Fig. 4) and on native U2 snRNPs (Fig. 5). In the mature 17S U2 snRNP, SF3a and SF3b splicing factors are bound to the 5′-terminal domain including stem/loops I, IIa, and IIb; Sm proteins are bound to the conserved Sm-binding sequence; and U2-specific proteins A′ and B“ are bound to stem/loop IV (62Perumal K. Sinha K. Henning D. Reddy R. J. Biol. 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Nucleic Acids Res. 1999; 27: 1025-1031Crossref PubMed Scopus (23) Google Scholar), and U2 snRNP particles are the substrates for U2 snRNA 3′ processing (34Kleinschmidt A.M. Pederson T. Mol. Cell. Biol. 1987; 7: 3131-3137Crossref PubMed Scopus (23) Google Scholar, 67Pederson T. Kleinschmidt A. Mol. Biol. Rep. 1990; 14: 179Crossref PubMed Scopus (0) Google Scholar, 68Wendelburg B.J. Marzluff W.F. Mol. Cell. Biol. 1992; 12: 4132-4141Crossref PubMed Scopus (5) Google Scholar). The activity of the human CCA-adding enzyme on native U2 snRNPs is consistent with the observation that 3′ maturation of U2 snRNA occursin vivo after snRNP assembly has begun.There are ample precedents for the ability of CCA-adding enzymes to recognize tRNA-like structures. tRNA consists of two structurally and functionally independent domains: a “top half” or minihelix consisting of the acceptor stem stacked on the TψC stem/loop, and a “bottom half” consisting of the DHU stem/loop stacked on the anticodon stem/loop (77Weiner A.M. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7383-7387Crossref PubMed Scopus (255) Google Scholar, 78Maizels N. Weiner A.M. Gesteland R.F. Cech T.R. Atkins J.F. The RNA World II. Cold Spring Harbor Press, Cold Spring Harbor, NY1999: 79-111Google Scholar). Not only does a tRNA minihelix serve as substrate for CCA-adding enzymes (12Shi P.Y. Weiner A.M. Maizels N. RNA (N. Y.). 1998; 4: 276-284PubMed Google Scholar, 57Hou Y.M. RNA (N. Y.). 2000; 6: 1031-1043Crossref PubMed Scopus (30) Google Scholar), but single-stranded RNA viruses often have 3′-terminal tRNA-like structures that serve as substrates for enzymes of tRNA metabolism including tRNA synthetases, the CCA-adding enzyme, and RNase P (11Giege R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12078-12081Crossref PubMed Scopus (36) Google Scholar, 79Fechter P. Giege R. Rudinger-Thirion J. J. Mol. Biol. 2001; 309: 387-399Crossref PubMed Scopus (20) Google Scholar, 80Mans R.M. Guerrier-Takada C. Altman S. Pleij C.W. Nucleic Acids Res. 1990; 18: 3479-3487Crossref PubMed Scopus (41) Google Scholar, 81Rao A.L. Dreher T.W. Marsh L.E. Hall T.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5335-5339Crossref PubMed Scopus (123) Google Scholar). Maize mitochondrial mRNAs also contain posttranscriptionally added 3′-terminal nucleotides that could be the product of a mitochondrial form of the CCA-adding enzyme (82Williams M.A. Johzuka Y. Mulligan R.M. Nucleic Acids Res. 2000; 28: 4444-4451Crossref PubMed Scopus (26) Google Scholar).Almost all U snRNAs have 3′-terminal stem/loops, presumably to confer stability on the RNAs (83Yu Y.T. Scharl E.C. Smith C.M. Steitz J.A. Gesteland R.F. Cech T.R. Atkins J.F. The RNA World II. Cold Spring Harbor Press, Cold Spring Harbor, NY1999: 487-524Google Scholar), and some small RNAs may be stabilized by posttranscription adenylation (45Sinha K.M. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 46Chen Y. Sinha K. Perumal K. Reddy R. RNA (N. Y.). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar). Although direct RNA sequencing data are scanty, the U2 snRNA 3′-terminal stem/loop appears to be followed by CCA only in mammals (human and rat); even frog U2 snRNA lacks CCA (Fig. 7). The U2 sequences of other organisms are either cDNA or DNA sequences. The cDNA sequences are all derived from poly(A)-tailed RNA, so that it is impossible to distinguish posttranscriptional 3′-terminal A addition from the poly(A) tail. The DNA sequences almost all have C+A-rich 3′-flanking regions; whether this C+A-rich region plays a role in 3′ end formation remains to be seen (20Ach R.A. Weiner A.M. Mol. Cell. Biol. 1987; 7: 2070-2079Crossref PubMed Scopus (33) Google Scholar, 25Cuello P. Boyd D.C. Dye M.J. Proudfoot N.J. Murphy S. EMBO J. 1999; 18: 2867-2877Crossref PubMed Scopus (50) Google Scholar, 38Huang Q. Pederson T. Nucleic Acids Res. 1999; 27: 1025-1031Crossref PubMed Scopus (23) Google Scholar), but it has already led to arbitrary assignment of U2 snRNA 3′ ends based solely on DNA sequence information. 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• W2039659797 date "2002-02-01" @default.
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• W2039659797 title "U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)" @default.
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• W2039659797 doi "https://doi.org/10.1074/jbc.m109559200" @default.
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