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W2022005363 abstract "The 3′-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853–6859). Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, we purified an adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed ∼75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3′-end adenylation of SRP RNA.AY029162 The 3′-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853–6859). Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, we purified an adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed ∼75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3′-end adenylation of SRP RNA. AY029162 signal recognition particle small nuclear RNA poly(A) polymerase polymerase chain reaction Most eukaryotic RNA molecules are synthesized as precursor molecules and are subsequently processed. These post-transcriptional processing events include 5′ capping, polyadenylation on the 3′ end of mRNAs, CCA turnover on the 3′ end of tRNAs, and splicing in the case of pre-mRNAs.Small RNAs are a diverse class of RNAs involved in a variety of important cellular functions (1Maxwell E.S. Fournier M.J. Annu. Rev. Biochem. 1995; 64: 897-934Crossref PubMed Scopus (536) Google Scholar, 2Steitz J.A. Black D.L. Gerke V. Parker K.A. Kramer A. Frendeway D. Keller W. Birnstiel M. Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles. Springer Verlag, Berlin1998: 115-154Google Scholar, 3Tollervey D. Kiss T. Curr. Opin. Cell Biol. 1997; 9: 337-342Crossref PubMed Scopus (375) Google Scholar, 4Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar). We recently showed that several small RNAs in human cells contain a single post-transcriptionally added adenylic acid residue on their 3′ ends. These RNAs include SRP1 RNA, 7SK RNA, U2 snRNA, and ribosomal 5S RNA (5Li W.Y. Reddy R. Henning D. Epstein P. Busch H. J. Biol. Chem. 1982; 257: 5136-5142Abstract Full Text PDF PubMed Google Scholar, 6O'Brien C.A. Wolin S.L. Genes Dev. 1994; 8: 2891-2903Crossref PubMed Scopus (183) Google Scholar, 7Reddy R. Henning D. Subrahmanyam C. Busch H. J. Biol. Chem. 1984; 259: 12265-12270Abstract Full Text PDF PubMed Google Scholar, 8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Ullu E. Weiner A.M. EMBO J. 1984; 3: 3303-3310Crossref PubMed Scopus (82) Google Scholar). SRP RNA is the RNA component of the signal recognition particle, which plays an important role in translocation of membrane proteins and secretory proteins (4Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar, 10Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). SRP RNA is synthesized in the nucleus by RNA polymerase III and is transported through the nucleolus on its way to the cytoplasm (11Jacobson M. Pederson T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7981-7986Crossref PubMed Scopus (126) Google Scholar). Data from our laboratory showed that SRP RNA in the nucleolus is already processed and adenylated on its 3′ end (12Chen Y. Sinha K. Perumal K. Gu J. Reddy R. J. Biol. Chem. 1999; 273: 35023-35031Abstract Full Text Full Text PDF Scopus (31) Google Scholar). These data indicate that the 3′-end processing and adenylation of SRP RNA are early events in the biogenesis of the signal recognition particle.The U2 snRNA is a required component for the splicing of pre-mRNA (13Black D.L. Chabot B. Steitz J.A. Cell. 1985; 42: 737-750Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 14Parker R. Siliciano P.G. Guthrie C. Cell. 1987; 49: 229-239Abstract Full Text PDF PubMed Scopus (323) Google Scholar). 60–70% of human SRP RNA, 7SK RNA, and U2 snRNA molecules contain post-transcriptionally added adenylic acid residues on their 3′ ends (8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In the case of ribosomal 5S RNA, ∼10% of the molecules was found to contain this post-transcriptionally added adenylic acid residue (8Sinha K. 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′-end adenylation of small RNAs is conserved through evolution because SRP RNA from rodents, amphibians, and insects contain this post-transcriptionally added adenylic acid residue (5Li W.Y. Reddy R. Henning D. Epstein P. Busch H. J. Biol. Chem. 1982; 257: 5136-5142Abstract Full Text PDF PubMed Google Scholar, 8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 15Perumal K Gu J. Reddy R. Mol. Cell. Biochem. 2000; 208: 99-109Crossref PubMed Google Scholar).The functions of this 3′-end adenylation found in the small RNAs are not fully understood. Because this 3′ adenylation occurs in many different RNAs with diverse functions, it is likely that the function of this 3′-adenylic acid residue is related to the metabolism of these RNAs. Data from our lab showed that small RNAs with post-transcriptionally added adenylic acid residue are not substrates for 3′ extension of RNAs by polyuridylation (16Chen Y. Sinha K. Perumal K. Reddy R. RNA ( N. Y. ). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar). In addition, the post-transcriptionally added 3′-adenylic acid residue has a relatively low turnover (8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These data suggest that one of the functions of this 3′ adenylation is to protect the 3′ ends of RNAs from digestion by exonucleases and also to prevent 3′ extensions by uridylation (16Chen Y. Sinha K. Perumal K. Reddy R. RNA ( N. Y. ). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar).Our initial studies were aimed at determining whether mRNA poly(A) polymerase (PAP) is responsible for this 3′ adenylation of small RNAs. The data showed that neither PAP nor tRNA nucleotidyltransferase is involved in carrying out this 3′ adenylation (17Sinha K. Perumal K. Chen Y. Reddy R. J. Biol. Chem. 1999; 274: 30826-30831Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). We carried out studies to identify and purify the enzyme responsible for post-transcriptional adenylation of human SRP RNA. This study shows the purification and characterization of a novel 3′-adenylating enzyme that has homology to PAP but it is a product of a distinct gene. A recombinant SRP RNA adenylating enzyme with adenylating activity was obtained and its activity compared with that of the SRP RNA adenylating enzyme purified from HeLa cells.DISCUSSIONThis study presents data on the purification of a new 3′-adenylating enzyme from HeLa cells. This purified enzyme is capable of adding adenylic acid residues to SRP/Alu RNA. The cDNA for this enzyme has been isolated, and interestingly, this enzyme is highly homologous to the well characterized mRNA PAP (Figs. 3 and 4). However, the SRP RNA adenylating enzyme is the product of a distinct gene located on chromosome 2.The homology between these two enzymes is ∼75% in the amino-terminal region (Figs. 3 and 4). However, the carboxyl terminus is highly divergent and shows only 25% homology between these two enzymes. It is not surprising that the amino terminus is conserved because this region contains the catalytic domain. The carboxyl terminus of the PAP contains the domain that interacts with other factors including the cleavage and polyadenylation specificity factor, which confers the specificity for the enzyme (28Murthy K.G. Manley J.L. Genes Dev. 1995; 9: 2672-2683Crossref PubMed Scopus (212) Google Scholar). Therefore, it is reasonable to expect divergence in the carboxyl terminus of the SRP RNA adenylating enzyme.The recombinant SRP RNA adenylating enzyme expressed in E. coli was capable of adenylating RNAs in vitro. However, there was no substrate specificity, and it added multiple adenylic acid residues (Fig. 7). This is not very surprising in light of what we already know about other purified enzymes. PAP in vivo and in nuclear extracts exhibits substrate specificity and adenylates only mRNAs containing the polyadenylation signal. However, purified PAP adenylates any RNA with a 3′-OH group (29Raabe T. Murthy K.G. Manley J.L. Mol. Cell. Biol. 1994; 14: 2946-2957Crossref PubMed Scopus (52) Google Scholar, 30Wahle E. Martin G. Schilts E. Keller W. EMBO J. 1991; 10: 4251-4257Crossref PubMed Scopus (90) Google Scholar). Similarly, the capping enzyme guanylyltransferase caps only RNAs transcribed by RNA polymerase II in vivo. However, purified guanylyltransferase caps any RNA with appropriate 5′ phosphates (31Martin S.A. Moss B. J. Biol. Chem. 1976; 251: 7313-7321Abstract Full Text PDF PubMed Google Scholar, 32Shuman S. J. Biol. Chem. 1982; 257: 7237-7245Abstract Full Text PDF PubMed Google Scholar). In all these cases, the specificity and regulation of enzymatic activity is caused by a multiprotein complex involved in these biological reactions. From the results obtained with the recombinant SRP RNA adenylating enzyme, it is very likely that other factors are necessary to carry out accurate and regulated adenylation in vitro. In this context, it is worth noting that the ribonucleoprotein complex consisting of Alu RNA bound to SRP 9/14 proteins is the required substrate for adenylation in vitro (17Sinha K. Perumal K. Chen Y. Reddy R. J. Biol. Chem. 1999; 274: 30826-30831Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Therefore, it may be necessary to use Alu RNA complexes with SRP 9/14 proteins to obtain accurate 3′-end adenylationin vitro. It is also possible that in addition to adenylating SRP RNA, this new adenylating enzyme is an mRNA poly(A) polymerase for some cellular mRNAs.The enzymatic activity of the SRP RNA adenylating enzyme may be regulated by other post-translational modifications such as phosphorylation. The PAP, for example, is highly phosphorylated and regulated through its phosphorylation (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Hyperphosphorylation of PAP inhibits adenylation activity, and the dephosphorylated enzyme is more active (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Several consensus and nonconsensus phosphorylation sites are present in the S/T-rich domain at the carboxyl terminus of the SRP RNA adenylating enzyme, and the corresponding region in PAP is highly phosphorylated. Therefore, it is very likely that the SRP RNA adenylating enzyme is phosphorylated in vivo, and its activity may be regulated by phosphorylation. Further work is needed to experimentally verify this possibility.The cDNA sequence for the SRP RNA adenylating enzyme is a partial sequence, and further work is needed to characterize the complete cDNA sequence in the 5′- and 3′-untranslated regions. The available 1,768 nucleotides of the 3′-untranslated region show no AAUAAA polyadenylation signal. Although the human PAP cDNA was isolated several years ago, the complete 5′-untranslated region has not been characterized, and the transcription initiation site is not yet known. Our unpublished data indicate that there is a long 5′-untranslated region in the case of the SRP RNA adenylating enzyme. It will be interesting to characterize the complete cDNA sequence of the PAP and SRP RNA adenylating enzyme because genomic organization (Fig. 5) suggests that the SRP RNA adenylating enzyme may have arisen by duplication of the PAP gene and subsequent divergence in the carboxyl terminus region. Most eukaryotic RNA molecules are synthesized as precursor molecules and are subsequently processed. These post-transcriptional processing events include 5′ capping, polyadenylation on the 3′ end of mRNAs, CCA turnover on the 3′ end of tRNAs, and splicing in the case of pre-mRNAs. Small RNAs are a diverse class of RNAs involved in a variety of important cellular functions (1Maxwell E.S. Fournier M.J. Annu. Rev. Biochem. 1995; 64: 897-934Crossref PubMed Scopus (536) Google Scholar, 2Steitz J.A. Black D.L. Gerke V. Parker K.A. Kramer A. Frendeway D. Keller W. Birnstiel M. Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles. Springer Verlag, Berlin1998: 115-154Google Scholar, 3Tollervey D. Kiss T. Curr. Opin. Cell Biol. 1997; 9: 337-342Crossref PubMed Scopus (375) Google Scholar, 4Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar). We recently showed that several small RNAs in human cells contain a single post-transcriptionally added adenylic acid residue on their 3′ ends. These RNAs include SRP1 RNA, 7SK RNA, U2 snRNA, and ribosomal 5S RNA (5Li W.Y. Reddy R. Henning D. Epstein P. Busch H. J. Biol. Chem. 1982; 257: 5136-5142Abstract Full Text PDF PubMed Google Scholar, 6O'Brien C.A. Wolin S.L. Genes Dev. 1994; 8: 2891-2903Crossref PubMed Scopus (183) Google Scholar, 7Reddy R. Henning D. Subrahmanyam C. Busch H. J. Biol. Chem. 1984; 259: 12265-12270Abstract Full Text PDF PubMed Google Scholar, 8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Ullu E. Weiner A.M. EMBO J. 1984; 3: 3303-3310Crossref PubMed Scopus (82) Google Scholar). SRP RNA is the RNA component of the signal recognition particle, which plays an important role in translocation of membrane proteins and secretory proteins (4Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar, 10Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). SRP RNA is synthesized in the nucleus by RNA polymerase III and is transported through the nucleolus on its way to the cytoplasm (11Jacobson M. Pederson T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7981-7986Crossref PubMed Scopus (126) Google Scholar). Data from our laboratory showed that SRP RNA in the nucleolus is already processed and adenylated on its 3′ end (12Chen Y. Sinha K. Perumal K. Gu J. Reddy R. J. Biol. Chem. 1999; 273: 35023-35031Abstract Full Text Full Text PDF Scopus (31) Google Scholar). These data indicate that the 3′-end processing and adenylation of SRP RNA are early events in the biogenesis of the signal recognition particle. The U2 snRNA is a required component for the splicing of pre-mRNA (13Black D.L. Chabot B. Steitz J.A. Cell. 1985; 42: 737-750Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 14Parker R. Siliciano P.G. Guthrie C. Cell. 1987; 49: 229-239Abstract Full Text PDF PubMed Scopus (323) Google Scholar). 60–70% of human SRP RNA, 7SK RNA, and U2 snRNA molecules contain post-transcriptionally added adenylic acid residues on their 3′ ends (8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In the case of ribosomal 5S RNA, ∼10% of the molecules was found to contain this post-transcriptionally added adenylic acid residue (8Sinha K. 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′-end adenylation of small RNAs is conserved through evolution because SRP RNA from rodents, amphibians, and insects contain this post-transcriptionally added adenylic acid residue (5Li W.Y. Reddy R. Henning D. Epstein P. Busch H. J. Biol. Chem. 1982; 257: 5136-5142Abstract Full Text PDF PubMed Google Scholar, 8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 15Perumal K Gu J. Reddy R. Mol. Cell. Biochem. 2000; 208: 99-109Crossref PubMed Google Scholar). The functions of this 3′-end adenylation found in the small RNAs are not fully understood. Because this 3′ adenylation occurs in many different RNAs with diverse functions, it is likely that the function of this 3′-adenylic acid residue is related to the metabolism of these RNAs. Data from our lab showed that small RNAs with post-transcriptionally added adenylic acid residue are not substrates for 3′ extension of RNAs by polyuridylation (16Chen Y. Sinha K. Perumal K. Reddy R. RNA ( N. Y. ). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar). In addition, the post-transcriptionally added 3′-adenylic acid residue has a relatively low turnover (8Sinha K. Gu J. Chen Y. Reddy R. J. Biol. Chem. 1998; 273: 6853-6859Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These data suggest that one of the functions of this 3′ adenylation is to protect the 3′ ends of RNAs from digestion by exonucleases and also to prevent 3′ extensions by uridylation (16Chen Y. Sinha K. Perumal K. Reddy R. RNA ( N. Y. ). 2000; 6: 1277-1288Crossref PubMed Scopus (40) Google Scholar). Our initial studies were aimed at determining whether mRNA poly(A) polymerase (PAP) is responsible for this 3′ adenylation of small RNAs. The data showed that neither PAP nor tRNA nucleotidyltransferase is involved in carrying out this 3′ adenylation (17Sinha K. Perumal K. Chen Y. Reddy R. J. Biol. Chem. 1999; 274: 30826-30831Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). We carried out studies to identify and purify the enzyme responsible for post-transcriptional adenylation of human SRP RNA. This study shows the purification and characterization of a novel 3′-adenylating enzyme that has homology to PAP but it is a product of a distinct gene. A recombinant SRP RNA adenylating enzyme with adenylating activity was obtained and its activity compared with that of the SRP RNA adenylating enzyme purified from HeLa cells. DISCUSSIONThis study presents data on the purification of a new 3′-adenylating enzyme from HeLa cells. This purified enzyme is capable of adding adenylic acid residues to SRP/Alu RNA. The cDNA for this enzyme has been isolated, and interestingly, this enzyme is highly homologous to the well characterized mRNA PAP (Figs. 3 and 4). However, the SRP RNA adenylating enzyme is the product of a distinct gene located on chromosome 2.The homology between these two enzymes is ∼75% in the amino-terminal region (Figs. 3 and 4). However, the carboxyl terminus is highly divergent and shows only 25% homology between these two enzymes. It is not surprising that the amino terminus is conserved because this region contains the catalytic domain. The carboxyl terminus of the PAP contains the domain that interacts with other factors including the cleavage and polyadenylation specificity factor, which confers the specificity for the enzyme (28Murthy K.G. Manley J.L. Genes Dev. 1995; 9: 2672-2683Crossref PubMed Scopus (212) Google Scholar). Therefore, it is reasonable to expect divergence in the carboxyl terminus of the SRP RNA adenylating enzyme.The recombinant SRP RNA adenylating enzyme expressed in E. coli was capable of adenylating RNAs in vitro. However, there was no substrate specificity, and it added multiple adenylic acid residues (Fig. 7). This is not very surprising in light of what we already know about other purified enzymes. PAP in vivo and in nuclear extracts exhibits substrate specificity and adenylates only mRNAs containing the polyadenylation signal. However, purified PAP adenylates any RNA with a 3′-OH group (29Raabe T. Murthy K.G. Manley J.L. Mol. Cell. Biol. 1994; 14: 2946-2957Crossref PubMed Scopus (52) Google Scholar, 30Wahle E. Martin G. Schilts E. Keller W. EMBO J. 1991; 10: 4251-4257Crossref PubMed Scopus (90) Google Scholar). Similarly, the capping enzyme guanylyltransferase caps only RNAs transcribed by RNA polymerase II in vivo. However, purified guanylyltransferase caps any RNA with appropriate 5′ phosphates (31Martin S.A. Moss B. J. Biol. Chem. 1976; 251: 7313-7321Abstract Full Text PDF PubMed Google Scholar, 32Shuman S. J. Biol. Chem. 1982; 257: 7237-7245Abstract Full Text PDF PubMed Google Scholar). In all these cases, the specificity and regulation of enzymatic activity is caused by a multiprotein complex involved in these biological reactions. From the results obtained with the recombinant SRP RNA adenylating enzyme, it is very likely that other factors are necessary to carry out accurate and regulated adenylation in vitro. In this context, it is worth noting that the ribonucleoprotein complex consisting of Alu RNA bound to SRP 9/14 proteins is the required substrate for adenylation in vitro (17Sinha K. Perumal K. Chen Y. Reddy R. J. Biol. Chem. 1999; 274: 30826-30831Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Therefore, it may be necessary to use Alu RNA complexes with SRP 9/14 proteins to obtain accurate 3′-end adenylationin vitro. It is also possible that in addition to adenylating SRP RNA, this new adenylating enzyme is an mRNA poly(A) polymerase for some cellular mRNAs.The enzymatic activity of the SRP RNA adenylating enzyme may be regulated by other post-translational modifications such as phosphorylation. The PAP, for example, is highly phosphorylated and regulated through its phosphorylation (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Hyperphosphorylation of PAP inhibits adenylation activity, and the dephosphorylated enzyme is more active (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Several consensus and nonconsensus phosphorylation sites are present in the S/T-rich domain at the carboxyl terminus of the SRP RNA adenylating enzyme, and the corresponding region in PAP is highly phosphorylated. Therefore, it is very likely that the SRP RNA adenylating enzyme is phosphorylated in vivo, and its activity may be regulated by phosphorylation. Further work is needed to experimentally verify this possibility.The cDNA sequence for the SRP RNA adenylating enzyme is a partial sequence, and further work is needed to characterize the complete cDNA sequence in the 5′- and 3′-untranslated regions. The available 1,768 nucleotides of the 3′-untranslated region show no AAUAAA polyadenylation signal. Although the human PAP cDNA was isolated several years ago, the complete 5′-untranslated region has not been characterized, and the transcription initiation site is not yet known. Our unpublished data indicate that there is a long 5′-untranslated region in the case of the SRP RNA adenylating enzyme. It will be interesting to characterize the complete cDNA sequence of the PAP and SRP RNA adenylating enzyme because genomic organization (Fig. 5) suggests that the SRP RNA adenylating enzyme may have arisen by duplication of the PAP gene and subsequent divergence in the carboxyl terminus region. This study presents data on the purification of a new 3′-adenylating enzyme from HeLa cells. This purified enzyme is capable of adding adenylic acid residues to SRP/Alu RNA. The cDNA for this enzyme has been isolated, and interestingly, this enzyme is highly homologous to the well characterized mRNA PAP (Figs. 3 and 4). However, the SRP RNA adenylating enzyme is the product of a distinct gene located on chromosome 2. The homology between these two enzymes is ∼75% in the amino-terminal region (Figs. 3 and 4). However, the carboxyl terminus is highly divergent and shows only 25% homology between these two enzymes. It is not surprising that the amino terminus is conserved because this region contains the catalytic domain. The carboxyl terminus of the PAP contains the domain that interacts with other factors including the cleavage and polyadenylation specificity factor, which confers the specificity for the enzyme (28Murthy K.G. Manley J.L. Genes Dev. 1995; 9: 2672-2683Crossref PubMed Scopus (212) Google Scholar). Therefore, it is reasonable to expect divergence in the carboxyl terminus of the SRP RNA adenylating enzyme. The recombinant SRP RNA adenylating enzyme expressed in E. coli was capable of adenylating RNAs in vitro. However, there was no substrate specificity, and it added multiple adenylic acid residues (Fig. 7). This is not very surprising in light of what we already know about other purified enzymes. PAP in vivo and in nuclear extracts exhibits substrate specificity and adenylates only mRNAs containing the polyadenylation signal. However, purified PAP adenylates any RNA with a 3′-OH group (29Raabe T. Murthy K.G. Manley J.L. Mol. Cell. Biol. 1994; 14: 2946-2957Crossref PubMed Scopus (52) Google Scholar, 30Wahle E. Martin G. Schilts E. Keller W. EMBO J. 1991; 10: 4251-4257Crossref PubMed Scopus (90) Google Scholar). Similarly, the capping enzyme guanylyltransferase caps only RNAs transcribed by RNA polymerase II in vivo. However, purified guanylyltransferase caps any RNA with appropriate 5′ phosphates (31Martin S.A. Moss B. J. Biol. Chem. 1976; 251: 7313-7321Abstract Full Text PDF PubMed Google Scholar, 32Shuman S. J. Biol. Chem. 1982; 257: 7237-7245Abstract Full Text PDF PubMed Google Scholar). In all these cases, the specificity and regulation of enzymatic activity is caused by a multiprotein complex involved in these biological reactions. From the results obtained with the recombinant SRP RNA adenylating enzyme, it is very likely that other factors are necessary to carry out accurate and regulated adenylation in vitro. In this context, it is worth noting that the ribonucleoprotein complex consisting of Alu RNA bound to SRP 9/14 proteins is the required substrate for adenylation in vitro (17Sinha K. Perumal K. Chen Y. Reddy R. J. Biol. Chem. 1999; 274: 30826-30831Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Therefore, it may be necessary to use Alu RNA complexes with SRP 9/14 proteins to obtain accurate 3′-end adenylationin vitro. It is also possible that in addition to adenylating SRP RNA, this new adenylating enzyme is an mRNA poly(A) polymerase for some cellular mRNAs. The enzymatic activity of the SRP RNA adenylating enzyme may be regulated by other post-translational modifications such as phosphorylation. The PAP, for example, is highly phosphorylated and regulated through its phosphorylation (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Hyperphosphorylation of PAP inhibits adenylation activity, and the dephosphorylated enzyme is more active (33Colgan D.F. Murthy K.G.K. Prives C. Manley J.L. Nature. 1996; 384: 282-285Crossref PubMed Scopus (140) Google Scholar). Several consensus and nonconsensus phosphorylation sites are present in the S/T-rich domain at the carboxyl terminus of the SRP RNA adenylating enzyme, and the corresponding region in PAP is highly phosphorylated. Therefore, it is very likely that the SRP RNA adenylating enzyme is phosphorylated in vivo, and its activity may be regulated by phosphorylation. Further work is needed to experimentally verify this possibility. The cDNA sequence for the SRP RNA adenylating enzyme is a partial sequence, and further work is needed to characterize the complete cDNA sequence in the 5′- and 3′-untranslated regions. The available 1,768 nucleotides of the 3′-untranslated region show no AAUAAA polyadenylation signal. Although the human PAP cDNA was isolated several years ago, the complete 5′-untranslated region has not been characterized, and the transcription initiation site is not yet known. Our unpublished data indicate that there is a long 5′-untranslated region in the case of the SRP RNA adenylating enzyme. It will be interesting to characterize the complete cDNA sequence of the PAP and SRP RNA adenylating enzyme because genomic organization (Fig. 5) suggests that the SRP RNA adenylating enzyme may have arisen by duplication of the PAP gene and subsequent divergence in the carboxyl terminus region." @default.
W2022005363 created "2016-06-24" @default.
W2022005363 creator A5065223265 @default.
W2022005363 creator A5080500061 @default.
W2022005363 creator A5082088286 @default.
W2022005363 creator A5087832026 @default.
W2022005363 date "2001-06-01" @default.
W2022005363 modified "2023-09-27" @default.
W2022005363 title "Purification, Characterization, and Cloning of the cDNA of Human Signal Recognition Particle RNA 3′-Adenylating Enzyme" @default.
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