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• W2053643897 abstract "The 3′-exonucleolytic decay of the mRNA for ribosomal protein S20 has been reconstituted in vitro using purified RNase II and crude extracts enriched for polynucleotide phosphorylase (PNPase) activity. We show that RNase II can catalyze the degradation of the 5′ two-thirds of the S20 mRNA and that prior oligoadenylation of the 3′ termini of truncated S20 mRNA substrates can significantly stimulate the initiation of degradation by RNase II. The intact S20 mRNA is, however, insensitive to attack by RNase II and polyadenylation of its 3′-end cannot overcome the natural resistance of the S20 mRNA to RNase II. Complete degradation of either the entire S20 mRNA without prior endonucleolytic cleavage or the 3′-terminal 147-residue fragment is dependent on both oligoadenylation and PNPase activity. Moreover, this process can take place in the absence of RNase E activity. Our data point to the importance of oligoadenylation in facilitating 3′-exonucleolytic activity and indicate that there are alternative degradative pathways. The implications for mRNA decay are discussed. The 3′-exonucleolytic decay of the mRNA for ribosomal protein S20 has been reconstituted in vitro using purified RNase II and crude extracts enriched for polynucleotide phosphorylase (PNPase) activity. We show that RNase II can catalyze the degradation of the 5′ two-thirds of the S20 mRNA and that prior oligoadenylation of the 3′ termini of truncated S20 mRNA substrates can significantly stimulate the initiation of degradation by RNase II. The intact S20 mRNA is, however, insensitive to attack by RNase II and polyadenylation of its 3′-end cannot overcome the natural resistance of the S20 mRNA to RNase II. Complete degradation of either the entire S20 mRNA without prior endonucleolytic cleavage or the 3′-terminal 147-residue fragment is dependent on both oligoadenylation and PNPase activity. Moreover, this process can take place in the absence of RNase E activity. Our data point to the importance of oligoadenylation in facilitating 3′-exonucleolytic activity and indicate that there are alternative degradative pathways. The implications for mRNA decay are discussed. INTRODUCTIONCurrent models for the turnover of mRNAs in Escherichia coli postulate that degradation is initiated by an endonucleolytic cleavage usually catalyzed by RNase E (1Mudd E.A. Krisch H.M. Higgins C.F. Mol. Microbiol. 1990; 4: 2127-2135Google Scholar, 2Babitzke P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1-5Google Scholar, 3Melefors Ö von Gabain A. Mol. Microbiol. 1991; 5: 857-864Google Scholar). Subsequent steps involve the attack on newly created 3′-ends by one or both of two exonucleases, RNase II or polynucleotide phosphorylase (PNPase) 1The abbreviation used is: PNPasepolynucleotide phosphorylase. (4Donovan W.P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 120-124Google Scholar, 5Belasco J.G. Higgins C.F. Gene (Amst.). 1988; 72: 15-23Google Scholar, 6Mclaren R.S. Newbury S.F. Dance G.S.C. Causton H.C. Higgins C.F. J. Mol. Biol. 1991; 221: 81-95Google Scholar). All three enzymes attack single-stranded RNAs (4Donovan W.P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 120-124Google Scholar, 5Belasco J.G. Higgins C.F. Gene (Amst.). 1988; 72: 15-23Google Scholar, 7Mackie G.A. J. Biol. Chem. 1992; 267: 1054-1061Google Scholar). This raises the question of how RNAs containing regions of extensive secondary structure can be degraded, since stem-loop structures can occlude potential RNase E sites (8Mackie G.A. Genereaux J.L. J. Mol. Biol. 1993; 234: 998-1012Google Scholar) and block the processive action of 3′-exonucleases (6Mclaren R.S. Newbury S.F. Dance G.S.C. Causton H.C. Higgins C.F. J. Mol. Biol. 1991; 221: 81-95Google Scholar). Recent work has implicated the products of the pcnB and pnp genes encoding poly(A) polymerase (9Cao G.J. Sarkar N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10380-10384Google Scholar, 10Masters M. Collums M.D. Oliver I.R. He L. MacNaughton E.J. Charters Y. J. Bacteriol. 1993; 175: 4405-4413Google Scholar) and PNPase, respectively, in the degradation of RNA I, a highly structured, small (108 nucleotides), untranslated, antisense inhibitor of the replication of colE1 replicons (11Xu F. Lin-Chao S. Cohen S.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6756-6760Google Scholar, 12Xu F. Cohen S.N. Nature. 1995; 374: 180-183Google Scholar). Polyadenylation also destabilizes the mRNA encoding ribosomal protein S15 (13Hajnsdorf E. Braun F. Haugel-Nielson J. Regnier P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3973-3977Google Scholar) and appears to regulate the decay of mRNAs generally (14O'Hara E.B. Chekanova J.A. Ingle C.A. Kushner Z.R. Peters E. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1807-1811Google Scholar). These results have prompted Cohen to propose that polyadenylation facilitates the exonucleolytic degradation of RNAs, particularly by PNPase, and that cleavage of RNAs mediated by RNase E is functionally coordinated with the 3′-exonucleolytic activity of PNPase (15Cohen S.N. Cell. 1995; 80: 829-832Google Scholar). The latter seems plausible in view of the association between these enzymes (16Carpousis A.J. van Houwe G. Ehretsmann C. Krisch H.M. Cell. 1994; 76: 889-900Google Scholar, 17Py B. Causton H. Mudd E.A. Higgins C.F. Mol. Microbiol. 1994; 14: 717-729Google Scholar).The mRNA for ribosomal protein S20 is a well characterized substrate for RNase E both in vivo and in vitro (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar, 19Rapaport L.R. Mackie G.A. J. Bacteriol. 1994; 176: 992-998Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). Both crude and purified RNase E cleave a synthetic S20 transcript (372 residues) to generate a number of products, the most prominent of which spans 147 residues coterminal with the substrate's 3′-end and is identical to a degradative intermediate found in vivo (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). Further degradation of the 3′-terminal 147-residue product of RNase E-mediated cleavage in vivo requires PNPase activity, since this product is stabilized and accumulates in pnp mutants (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar). The 147-residue product is, however, essentially stable in vitro, even in relatively crude extracts (8Mackie G.A. Genereaux J.L. J. Mol. Biol. 1993; 234: 998-1012Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). The S20 mRNA has served as a model to search for conditions that would permit its total degradation in vitro and to test some of the predictions of Cohen's model (15Cohen S.N. Cell. 1995; 80: 829-832Google Scholar). We show here that RNase II can participate in the degradation in vitro of the 5′ two-thirds of the S20 mRNA and that oligoadenylation can significantly stimulate the activity of RNase II. Furthermore, oligoadenylation is a prerequisite for the complete degradation of the S20 mRNA, a process also dependent on PNPase, but both steps can be completely uncoupled from endonucleolytic cleavage by RNase E. INTRODUCTIONCurrent models for the turnover of mRNAs in Escherichia coli postulate that degradation is initiated by an endonucleolytic cleavage usually catalyzed by RNase E (1Mudd E.A. Krisch H.M. Higgins C.F. Mol. Microbiol. 1990; 4: 2127-2135Google Scholar, 2Babitzke P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1-5Google Scholar, 3Melefors Ö von Gabain A. Mol. Microbiol. 1991; 5: 857-864Google Scholar). Subsequent steps involve the attack on newly created 3′-ends by one or both of two exonucleases, RNase II or polynucleotide phosphorylase (PNPase) 1The abbreviation used is: PNPasepolynucleotide phosphorylase. (4Donovan W.P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 120-124Google Scholar, 5Belasco J.G. Higgins C.F. Gene (Amst.). 1988; 72: 15-23Google Scholar, 6Mclaren R.S. Newbury S.F. Dance G.S.C. Causton H.C. Higgins C.F. J. Mol. Biol. 1991; 221: 81-95Google Scholar). All three enzymes attack single-stranded RNAs (4Donovan W.P. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 120-124Google Scholar, 5Belasco J.G. Higgins C.F. Gene (Amst.). 1988; 72: 15-23Google Scholar, 7Mackie G.A. J. Biol. Chem. 1992; 267: 1054-1061Google Scholar). This raises the question of how RNAs containing regions of extensive secondary structure can be degraded, since stem-loop structures can occlude potential RNase E sites (8Mackie G.A. Genereaux J.L. J. Mol. Biol. 1993; 234: 998-1012Google Scholar) and block the processive action of 3′-exonucleases (6Mclaren R.S. Newbury S.F. Dance G.S.C. Causton H.C. Higgins C.F. J. Mol. Biol. 1991; 221: 81-95Google Scholar). Recent work has implicated the products of the pcnB and pnp genes encoding poly(A) polymerase (9Cao G.J. Sarkar N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10380-10384Google Scholar, 10Masters M. Collums M.D. Oliver I.R. He L. MacNaughton E.J. Charters Y. J. Bacteriol. 1993; 175: 4405-4413Google Scholar) and PNPase, respectively, in the degradation of RNA I, a highly structured, small (108 nucleotides), untranslated, antisense inhibitor of the replication of colE1 replicons (11Xu F. Lin-Chao S. Cohen S.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6756-6760Google Scholar, 12Xu F. Cohen S.N. Nature. 1995; 374: 180-183Google Scholar). Polyadenylation also destabilizes the mRNA encoding ribosomal protein S15 (13Hajnsdorf E. Braun F. Haugel-Nielson J. Regnier P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3973-3977Google Scholar) and appears to regulate the decay of mRNAs generally (14O'Hara E.B. Chekanova J.A. Ingle C.A. Kushner Z.R. Peters E. Kushner S.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1807-1811Google Scholar). These results have prompted Cohen to propose that polyadenylation facilitates the exonucleolytic degradation of RNAs, particularly by PNPase, and that cleavage of RNAs mediated by RNase E is functionally coordinated with the 3′-exonucleolytic activity of PNPase (15Cohen S.N. Cell. 1995; 80: 829-832Google Scholar). The latter seems plausible in view of the association between these enzymes (16Carpousis A.J. van Houwe G. Ehretsmann C. Krisch H.M. Cell. 1994; 76: 889-900Google Scholar, 17Py B. Causton H. Mudd E.A. Higgins C.F. Mol. Microbiol. 1994; 14: 717-729Google Scholar).The mRNA for ribosomal protein S20 is a well characterized substrate for RNase E both in vivo and in vitro (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar, 19Rapaport L.R. Mackie G.A. J. Bacteriol. 1994; 176: 992-998Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). Both crude and purified RNase E cleave a synthetic S20 transcript (372 residues) to generate a number of products, the most prominent of which spans 147 residues coterminal with the substrate's 3′-end and is identical to a degradative intermediate found in vivo (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). Further degradation of the 3′-terminal 147-residue product of RNase E-mediated cleavage in vivo requires PNPase activity, since this product is stabilized and accumulates in pnp mutants (18Mackie G.A. J. Bacteriol. 1989; 171: 4112-4120Google Scholar). The 147-residue product is, however, essentially stable in vitro, even in relatively crude extracts (8Mackie G.A. Genereaux J.L. J. Mol. Biol. 1993; 234: 998-1012Google Scholar, 20Mackie G.A. J. Bacteriol. 1991; 173: 2488-2497Google Scholar). The S20 mRNA has served as a model to search for conditions that would permit its total degradation in vitro and to test some of the predictions of Cohen's model (15Cohen S.N. Cell. 1995; 80: 829-832Google Scholar). We show here that RNase II can participate in the degradation in vitro of the 5′ two-thirds of the S20 mRNA and that oligoadenylation can significantly stimulate the activity of RNase II. Furthermore, oligoadenylation is a prerequisite for the complete degradation of the S20 mRNA, a process also dependent on PNPase, but both steps can be completely uncoupled from endonucleolytic cleavage by RNase E." @default.
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• W2053643897 title "Differential Sensitivities of Portions of the mRNA for Ribosomal Protein S20 to 3′-Exonucleases Dependent on Oligoadenylation and RNA Secondary Structure" @default.
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