Role of Bacillus subtilis RNase J1 Endonuclease and 5′-Exonuclease Activities in trp Leader RNA Turnover
2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m801461200
ISSN1083-351X
AutoresGintaras Deikus, Ciarán Condon, David H. Bechhofer,
Tópico(s)Bacteriophages and microbial interactions
ResumoThe 140-nucleotide trp leader RNA, which is formed by transcription termination under conditions of high intracellular tryptophan, was used to study RNA turnover in Bacillus subtilis. We showed in vivo that the amount of endonuclease cleavage at ∼nucleotide 100 is decreased under conditions where RNase J1 concentration is reduced. In addition, under these conditions the level of 3′-terminal RNA fragments, which contain the strong transcription terminator structure, increases dramatically. These results implicated RNase J1 in the initiation of trp leader RNA decay as well as in the subsequent steps leading to complete turnover of the terminator fragment. To confirm a direct role for RNase J1, experiments were performed in vitro with various forms of trp leader RNA and 3′-terminal RNA fragments. Specific endonuclease cleavages, which were restricted to single-stranded regions not bound by protein, were observed. Degradation of the 3′-terminal fragment by the 5′ to 3′-exonuclease activity of RNase J1 was also demonstrated, although the presence of strong secondary structure impeded RNase J1 processivity to some extent. These results are consistent with a model for mRNA decay in Bacillus subtilis whereby the downstream products of RNase J1 endonucleolytic cleavage become substrates for the 5′ to 3′-exoribonuclease activity of the enzyme. The 140-nucleotide trp leader RNA, which is formed by transcription termination under conditions of high intracellular tryptophan, was used to study RNA turnover in Bacillus subtilis. We showed in vivo that the amount of endonuclease cleavage at ∼nucleotide 100 is decreased under conditions where RNase J1 concentration is reduced. In addition, under these conditions the level of 3′-terminal RNA fragments, which contain the strong transcription terminator structure, increases dramatically. These results implicated RNase J1 in the initiation of trp leader RNA decay as well as in the subsequent steps leading to complete turnover of the terminator fragment. To confirm a direct role for RNase J1, experiments were performed in vitro with various forms of trp leader RNA and 3′-terminal RNA fragments. Specific endonuclease cleavages, which were restricted to single-stranded regions not bound by protein, were observed. Degradation of the 3′-terminal fragment by the 5′ to 3′-exonuclease activity of RNase J1 was also demonstrated, although the presence of strong secondary structure impeded RNase J1 processivity to some extent. These results are consistent with a model for mRNA decay in Bacillus subtilis whereby the downstream products of RNase J1 endonucleolytic cleavage become substrates for the 5′ to 3′-exoribonuclease activity of the enzyme. The number of documented ribonuclease activities in Bacillus subtilis has grown rapidly in the last few years, with at least 15 enzymes known (1Condon C. Curr. Opin. Microbiol. 2007; 10: 271-278Crossref PubMed Scopus (120) Google Scholar). Of those that have been identified recently, perhaps the most relevant ribonuclease for RNA turnover is RNase J1, encoded by the rnjA (formerly ykqC) gene. RNase J1 was originally identified as an endoribonuclease that shares characteristics with Escherichia coli RNase E (2Even S. Pellegrini O. Zig L. Labas V. Vinh J. Brechemmier-Baey D. Putzer H. Nucleic Acids Res. 2005; 33: 2141-2152Crossref PubMed Scopus (249) Google Scholar), suggesting that RNase J1 may be the primary nuclease activity responsible for regulating mRNA decay in B. subtilis as RNase E is thought to be in E. coli (3Coburn G.A. Mackie G.A. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 55-5108Crossref PubMed Scopus (267) Google Scholar, 4Kushner S.R. J. Bacteriol. 2002; 184: 4658-4665Crossref PubMed Scopus (198) Google Scholar). Indeed, Putzer and co-workers (2Even S. Pellegrini O. Zig L. Labas V. Vinh J. Brechemmier-Baey D. Putzer H. Nucleic Acids Res. 2005; 33: 2141-2152Crossref PubMed Scopus (249) Google Scholar) reported that global mRNA half-life was increased slightly in an RNase J1 mutant that was grown under conditions where RNase J1 was depleted. More recently, the surprising finding was reported that RNase J1 also has 5′ to 3′-exoribonuclease activity (5Mathy N. Benard L. Pellegrini O. Daou R. Wen T. Condon C. Cell. 2007; 129: 681-692Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), an activity not previously known to exist in bacteria. The presence of a 5′ to 3′-exoribonuclease activity in B. subtilis provides a rationale for the often observed 5′-end dependence of mRNA decay in B. subtilis (6Condon C. Microbiol. Mol. Biol. Rev. 2003; 67: 157-174Crossref PubMed Scopus (126) Google Scholar). RNase J1 has also been shown to be important for processing of two stable RNAs, scRNA (7Yao S. Blaustein J.B. Bechhofer D.H. Nucleic Acids Res. 2007; 35: 4464-4473Crossref PubMed Scopus (35) Google Scholar) and 16S rRNA (8Britton R.A. Wen T. Schaefer L. Pellegrini O. Uicker W.C. Mathy N. Tobin C. Daou R. Szyk J. Condon C. Mol. Microbiol. 2007; 63: 127-138Crossref PubMed Scopus (117) Google Scholar). Another endoribonuclease, RNase J2, encoded by the rnjB (formerly ymfA) gene, shares characteristics with RNase J1 (2Even S. Pellegrini O. Zig L. Labas V. Vinh J. Brechemmier-Baey D. Putzer H. Nucleic Acids Res. 2005; 33: 2141-2152Crossref PubMed Scopus (249) Google Scholar); however, whereas RNase J1 is essential, RNase J2 is not. RNase J1 activity is sensitive to the phosphorylation state of the 5′-end, with severalfold greater activity observed with monophosphorylated or hydroxylated 5′-ends than with triphosphorylated 5′-ends (2Even S. Pellegrini O. Zig L. Labas V. Vinh J. Brechemmier-Baey D. Putzer H. Nucleic Acids Res. 2005; 33: 2141-2152Crossref PubMed Scopus (249) Google Scholar, 5Mathy N. Benard L. Pellegrini O. Daou R. Wen T. Condon C. Cell. 2007; 129: 681-692Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 9de la Sierra-Gallay I.L. Zig L. Jamalli A. Putzer H. Nat. Struct. Mol. Biol. 2008; 15: 206-212Crossref PubMed Scopus (142) Google Scholar). E. coli RNase E also shows a strong preference for monophosphorylated versus triphosphorylated 5′-ends, but, unlike RNase J1, it is poorly active on a hydroxylated 5′-end (10Mackie G.A. Nature. 1998; 395: 720-723Crossref PubMed Scopus (341) Google Scholar, 11Jiang X. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9211-9216Crossref PubMed Scopus (104) Google Scholar). It has been suggested that the 5′-end-dependent action of RNase E on E. coli RNAs is preceded by a pyrophosphatase activity that converts the 5′-triphosphate end of the transcription product to a 5′-monophosphate end (12Celesnik H. Deana A. Belasco J.G. Mol. Cell. 2007; 27: 79-90Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The activity in E. coli responsible for this conversion has been identified recently as the pyrophosphohydrolase RppH (formerly NudH) (13Deana A. Celesnik H. Belasco J.G. Nature. 2008; 451: 355-358Crossref PubMed Scopus (318) Google Scholar). No such enzyme has yet been identified in B. subtilis. To study RNA processing and turnover in B. subtilis, we have been using the 140-nucleotide (nt) 2The abbreviations used are: nt, nucleotide; TRAP, trp RNA-binding attenuation protein; pCp, cytidine 3′,5′-bis(phosphate),[5′-32P]; IPTG, isopropyl-1-thio-β-d-galactopyranoside. 2The abbreviations used are: nt, nucleotide; TRAP, trp RNA-binding attenuation protein; pCp, cytidine 3′,5′-bis(phosphate),[5′-32P]; IPTG, isopropyl-1-thio-β-d-galactopyranoside. trp leader RNA as a model. The B. subtilis trp operon is regulated at the level of transcription termination/antitermination (14Babitzke P. Gollnick P. J. Bacteriol. 2001; 183: 5795-5802Crossref PubMed Scopus (72) Google Scholar, 15Henkin T.M. Yanofsky C. BioEssays. 2002; 24: 700-707Crossref PubMed Scopus (206) Google Scholar), which is controlled by binding of the trp RNA-binding attenuation protein (TRAP, encoded by the mtrB gene) to trp leader RNA. In the absence of TRAP binding, when intracellular tryptophan concentration is low, an antiterminator structure forms (Fig. 1A) that precludes formation of the terminator structure and transcription proceeds into the trp structural genes, which encode tryptophan biosynthetic enzymes. When the intracellular supply of tryptophan is sufficient, the 11-mer TRAP protein complex is activated by binding of tryptophan, allowing the complex to bind to 11 trinucleotide repeats (GAG or UAG) located between nt 36–91 of the trp leader sequence (Fig. 1B). TRAP binding results in formation of a stem-loop structure (nt 108–133) that induces transcription termination, generating the 140-nt trp leader RNA. The small size of this RNA makes it a useful tool for studying in detail the activity of ribonucleases in vivo and in vitro. We have recently shown that initiation of trp leader RNA decay is by an endonuclease cleavage at ∼nt 100 of trp leader RNA (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Turnover of the 5′-cleavage fragment, which is essential for release and ongoing activity of TRAP, is mediated by polynucleotide phosphorylase (PNPase), a phosphorolytic 3′ to 5′-exoribonuclease (17Deikus G. Babitzke P. Bechhofer D.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2747-2751Crossref PubMed Scopus (38) Google Scholar). Here we show that the endonuclease cleavage at nt 100 is RNase J1-dependent in vivo and that cleavage at this site, as well as 5′ to 3′-exonucleolytic degradation of the resulting 3′-terminal fragment, is catalyzed directly by RNase J1 in vitro. Bacterial Strains—The "wild-type" B. subtilis strain for in vivo experiments was BG581, which is trpC2 thr-5 and carries plasmid pGD5, a high copy number plasmid that contains the trp leader region and the mtrB gene (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). For experiments with RNase J1 and J2 mutants, BG581 was transformed with pMAP65, which carries the lacI gene (18Petit M.A. Dervyn E. Rose M. Entian K.D. McGovern S. Ehrlich S.D. Bruand C. Mol. Microbiol. 1998; 29: 261-273Crossref PubMed Scopus (131) Google Scholar), to give BG598. BG598 was transformed with chromosomal DNA isolated from the RNase J1 conditional mutant and the RNase J2 deletion mutant described previously (8Britton R.A. Wen T. Schaefer L. Pellegrini O. Uicker W.C. Mathy N. Tobin C. Daou R. Szyk J. Condon C. Mol. Microbiol. 2007; 63: 127-138Crossref PubMed Scopus (117) Google Scholar) to give BG600 (RNase J1 mutant) and BG601 (RNase J2 mutant). Purification of RNase J1 and H76A Mutant Protein—Cloning and overexpression of the rnjA gene, as well as the H76A mutant derivative, were as described (8Britton R.A. Wen T. Schaefer L. Pellegrini O. Uicker W.C. Mathy N. Tobin C. Daou R. Szyk J. Condon C. Mol. Microbiol. 2007; 63: 127-138Crossref PubMed Scopus (117) Google Scholar). Preparation of RNA Substrates—trp leader RNA was synthesized using T7 RNA polymerase transcription (Ambion T7 MAXIscript kit) of a PCR fragment that contained the trp leader sequence with a T7 promoter included in the upstream primer. The transcription product was purified from a 6% denaturing polyacrylamide gel as described (19Alberta J.A. Rundell K. Stiles C.D. J. Biol. Chem. 1994; 269: 4532-4538Abstract Full Text PDF PubMed Google Scholar), using a diffusion buffer that contained 1 m ammonium acetate, 2 mm magnesium acetate, 1 mm EDTA, and 0.2% SDS. Quantitation of unlabeled trp leader RNA was done spectrophotometrically. For 5′-triphosphate end labeling of trp leader RNA, transcription was done in the presence of [γ-32P]GTP. For 5′-monophosphate end labeling of trp leader RNA, cold trp leader RNA was treated with calf intestine alkaline phosphatase (New England Biolabs) and labeled with polynucleotide kinase using [γ-32P]ATP. For 3′-end labeling, cytidine 3′,5′-bis(phosphate),[5′-32P] (referred to as "pCp") (New England Nuclear) was ligated to the 3′-end of trp leader RNA using T4 RNA ligase (New England Biolabs) for 6 h at 18 °C. Labeled RNA was purified from a 6% denaturing polyacrylamide gel as above. RNA oligonucleotides, bearing a 5′-hydroxyl end, were obtained from Integrated DNA Technologies and were labeled at the 5′-end (monophosphate) with polynucleotide kinase and at the 3′-end by T4 RNA ligase, as above for full-length trp leader RNA, and were purified from 9% denaturing polyacrylamide gels. For the RNA oligonucleotide with the labeled triphosphate end, RNA was synthesized by T7 RNA polymerase transcription in the presence of [γ-32P]GTP and purified from a 9% polyacrylamide gel. Assay of RNase J1 Activity—Reactions were done using 10 nm RNA substrate, of which 10% was labeled, and 1.4 μm RNase J1. The buffer contained 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.1 mm dithiothreitol, 5 mm MgCl2. For analysis of full-length trp leader RNA, 2.5 mm tryptophan was present, with or without 37.5 nm TRAP. For experiments where TRAP was present, RNA was incubated with TRAP for 15 min at 22 °C before addition of RNase J1. Initially, incubation with RNase J1 was done at two temperatures, 22 and 37 °C. However, no difference was observed in the results, so subsequent experiments were performed at 22 °C. The reactions were stopped by addition of EDTA to 10 mm and an equal volume of gel loading buffer (Ambion) and were put immediately on dry ice. Samples were loaded on either an 8 or a 22.5% denaturing polyacrylamide gel. For thin layer chromatography, reactions were stopped by addition of 10 mm EDTA and aliquots were applied to a polyethyleneimine-cellulose sheet (Merck) and developed in 1 m LiCl. Quantitation of decay products was done with a Storm 860 PhosphorImager (GE Healthcare) or a Typhoon TRIO variable mode imager (GE Healthcare). PNPase Binding Assay—Binding of PNPase to the 3′-end of RNA oligonucleotides was performed as described (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Northern Blot Analysis—RNA isolation from B. subtilis strains grown in 2× YT (1% yeast extract, 2% tryptone, 1% NaCl) and Northern blot analysis were performed as described (17Deikus G. Babitzke P. Bechhofer D.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2747-2751Crossref PubMed Scopus (38) Google Scholar). To control for RNA loading in Northern blot analyses, membranes were stripped and probed for 5S rRNA as described (20Sharp J.S. Bechhofer D.H. J. Bacteriol. 2003; 185: 5372-5379Crossref PubMed Scopus (55) Google Scholar). High resolution Northern blot analysis was performed using a 7% denaturing polyacrylamide gel and electroblotting at 20 volts overnight, followed by an additional 60 min at 30 volts. 5′-end-labeled oligonucleotides were used as probes, except for the ΔermC riboprobe (20Sharp J.S. Bechhofer D.H. J. Bacteriol. 2003; 185: 5372-5379Crossref PubMed Scopus (55) Google Scholar) used for the Northern blot in Fig. 7. We had shown previously that endonuclease cleavage around nt 100 of trp leader RNA was responsible for initiation of decay (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To study whether RNase J1 was involved in trp leader RNA turnover, strains were constructed that carried the high copy number plasmid pGD5 (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) containing the trp leader region and a copy of the mtrB gene and that conditionally expressed RNase J1 under control of the IPTG-inducible pspac promoter or had a deletion of the gene encoding RNase J2. Expression of the chromosomal copy of the trp leader is low, and the presence of pGD5 results in a 30-fold increase in trp leader RNA levels, allowing easier detection of processing products. To ensure low level expression of RNase J1 in the absence of IPTG, the strains also contained pMAP65, a plasmid that carries the lacI gene, providing extra copies of the lac repressor (18Petit M.A. Dervyn E. Rose M. Entian K.D. McGovern S. Ehrlich S.D. Bruand C. Mol. Microbiol. 1998; 29: 261-273Crossref PubMed Scopus (131) Google Scholar). Northern blot analysis of trp leader RNA was performed using a probe that was complementary to sequences upstream of the endonuclease cleavage site (probe 1 in Fig. 1B). The results (Fig. 2A) showed that there was a 3-fold (average of five experiments) decrease in the ∼100-nt fragment when the RNase J1 conditional strain was grown in the absence of IPTG compared with the presence of IPTG. Additional faint bands were detected above the 100-nt band. These are likely minor alternative endonuclease cleavage products or the result of exonuclease decay of full-length RNA. Deletion of the RNase J2-encoding gene had no effect on trp leader RNA processing (Fig. 2A) (For unknown reasons, we consistently observed a lower quantity of trp leader RNA in the RNase J2 deletion strain. However, the amount of ∼100-nt fragment relative to full-length trp leader RNA was virtually the same in the RNase J2 deletion strain and the wild-type strain.) A high resolution Northern blot analysis using probe 2 showed that the major endonuclease cleavages occurred at about nt 100–102 (Fig. 2B). Northern blot analysis was also done using a probe that was complementary to the stem-loop terminator sequence (probe 3 in Fig. 1B). In this case, the results showed a large increase (∼20-fold) in the amount of small RNA fragments ranging in size from ∼30–40 nt (Fig. 2C; the stem-loop structure itself is 26 nt). Probing of a high resolution Northern blot with probe 3 showed two clusters of fragments from about 32–36 and 38–42 nt (Fig. 2D). The increased level of 3′-terminal fragments observed in Fig. 2C is consistent with these fragments being generated by endonuclease cleavages at ∼nt 100 and resisting further decay in conditions of reduced RNase J1. These results suggested that the endonuclease activity of RNase J1 played a role in the initiation of trp leader RNA decay and that the exonuclease and/or endonuclease activity of RNase J1 was important in the subsequent degradation of the 3′-terminal fragment. We therefore sought to characterize RNase J1 activity on trp leader RNA in vitro to determine whether the observed in vivo effects could be attributed directly to RNase J1. trp leader RNA transcripts were prepared to contain a labeled triphosphate 5′-end, which was incubated with RNase J1 in the presence or absence of purified TRAP protein. (Note that to ensure efficient transcription by T7 RNA polymerase the in vitro transcripts contained three extra Gs at the 5′-end. Thus, 5′-end-containing fragments observed in vitro are 3 nucleotides longer than the corresponding fragments in vivo. Numbering of the fragment sizes in Fig. 3 is according to the numbering of the natural trp leader RNA as shown in Fig. 1.) As a control, labeled RNAs were also incubated with the RNase J1 H76A mutant protein, which has severely reduced nuclease activity (8Britton R.A. Wen T. Schaefer L. Pellegrini O. Uicker W.C. Mathy N. Tobin C. Daou R. Szyk J. Condon C. Mol. Microbiol. 2007; 63: 127-138Crossref PubMed Scopus (117) Google Scholar). The results in Fig. 3, A and B, showed that trp leader RNA was indeed cleaved endonucleolytically by RNase J1 and that this cleavage occurred differently depending on the presence or absence of TRAP. In the absence of TRAP, RNase J1 cleaved broadly in the single-stranded region between the 5′-stem-loop structure and the antiterminator structure (nt 33–59; see Fig. 1A). The sizes of the ladder of fragments seen in the high resolution ("sequencing") gel in Fig. 3B, bottom lane 2, corresponded well with multiple endonuclease cleavages in this single-stranded region. In the presence of TRAP, however, cleavage was observed at ∼nt 100 as well as in a smaller number of sites in the region between the 5′-stem-loop and the beginning of the TRAP binding site (Fig. 3A and 3B, lane 4). Virtually no cleavage activity was observed with the H76A mutant protein. The rate of cleavage appeared to be slower in the presence of TRAP, and this is likely due to binding of TRAP to sites that cover 40% of the RNA sequence, thus reducing the available number of cleavage sites. Also, note that in addition to the products of RNase J1 activity that are visible on the gel in Fig. 3A, other small endonuclease fragments, as well as a low level of single nucleotide (as determined by thin layer chromatography), are produced (data not shown). When trp leader RNA was synthesized to contain a 5′-monophosphate end (Fig. 3C), the relative amount of endonuclease cleavage was reduced by about half (Fig. 3D). Also, in the absence of TRAP, cleavage at a distal site (at about 54 nt) was preferred, unlike the broad range of cleavages in this region seen with the triphosphorylated substrate (Fig. 3A and see "Discussion"). trp leader RNA transcripts (i.e. bearing a 5′-triphosphate) were also labeled at the 3′-end by addition of [32P]pCp (see "Experimental Procedures") in order to observe the generation of 3′-end-containing fragments. In this case, the 5′-exonuclease activity of RNase J1 made it difficult to assess a rate or exact site of initial cleavage, because downstream products of endonuclease cleavage were subsequently degraded from their 5′-ends. Nevertheless, the approximate location of endonuclease cleavages could be determined by short incubation with RNase J1 and resolution of the products on a sequencing gel (Fig. 4A, lanes 2 and 4). (Note that the numbering in Fig. 4A takes into account the extra nucleotide added by addition of pCp and is according to the numbering of the natural trp leader RNA shown in Fig. 1.) The strongest cleavages in the absence of TRAP were at nt 35, which is a few nucleotides after the 5′-stem-loop, and at nt 51, which is several nucleotides upstream of the antiterminator stem. A cleavage at nt 99 was also observed, which is in a bulged region on the downstream side of the antiterminator stem. Additional cleavages may have occurred closer to the 3′-end, but these were not detected on this gel. In the presence of TRAP, there was a cleavage at nt 35 and a weak cleavage at nt 51, but the stronger cleavages were in the single-stranded region immediately downstream of the TRAP binding site. The results with the 3′-labeled substrate were consistent with the cleavages observed with the 5′-labeled substrate (Fig. 3). The ladder seen between nt 35 and 54 with the 5′-labeled RNA in the absence of TRAP (Fig. 3B) is replaced in the case of the 3′-labeled RNA with more discrete species at the boundaries of this zone (nt 35 to 51). This suggests that the species generated by endonucleolytic cleavage are rapidly nibbled down to nt 51 by the 5′ to 3′-exonuclease activity of RNase J1. If the in vitro results obtained with TRAP-bound RNA reflected events occurring in vivo, we predicted that, in a strain grown in the presence of tryptophan, RNase J1 would cleave in the single-stranded regions of trp leader RNA on either side of the TRAP binding site (nt 36–91), generating an RNA fragment about the size of this region (56 nt). We probed for such a fragment using probe 2 (Fig. 1B). As can be seen in the high resolution Northern blot in Fig. 4B, a doublet of ∼56–57 nt was detected by this probe. This fragment was not detected by the upstream probe 1 (data not shown) and so is likely to consist of the TRAP binding region. Thus, the observed RNase J1 cleavages in vitro appear to take place in vivo, suggesting that this enzyme is directly responsible for decay-initiating RNA turnover. The observed effect of reduced RNase J1 levels on 3′-terminal fragment turnover (Fig. 2C) prompted an in vitro investigation of RNase J1 activity on this RNA. An RNA oligonucleotide, designated "3′-stem," was used that contained the sequence of the 3′-end of trp leader RNA (nt 99–140; Fig. 1C). As controls, two other RNAs were used, "3′-no-stem," which was similar to 3′-stem RNA except that six of the complementary nucleotides in the downstream side of the stem were changed such that the strong secondary structure could not form, and "3′-stem+5′-ext," which had the same sequence as 3′-stem RNA plus a 24-nt extension added at the 5′-end (Fig. 1C). (For technical reasons, 3′-stem+5′-ext RNA had only two nucleotides downstream of the stem structure rather than the six nucleotides in 3′-stem RNA. We assumed this difference was irrelevant under in vitro conditions where no 3′ to 5′-exonuclease was present.) As a test of whether the predicted structure was forming at the 3′-end of these RNA oligonucleotides, PNPase binding experiments were done. We have shown previously that PNPase binds well to substrates with 3′-single-stranded tails but binds poorly to substrates with 3′-terminal secondary structure (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). From the binding assay shown in Fig. 1D, it was clear that the level of PNPase binding to 3′-stem RNA and 3′-stem+5′-ext RNA was much lower than the level of binding to 3′-no-stem RNA. Quantitation of the results with 2.5 nm PNPase showed that 80% of 3′-no-stem RNA was bound as opposed to only 15% of 3′-stem and 3′-stem+5′-ext RNA. These percentages are consistent with our previous data on binding of PNPase to trp leader RNA without TRAP present (single-stranded RNA at 3′-end) and with TRAP present (secondary structure at 3′-end) (16Deikus G. Bechhofer D.H. J. Biol. Chem. 2007; 282: 20238-20244Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Thus, although it is likely that the RNA oligonucleotide substrates assume alternative conformations to some extent, the PNPase binding data suggest that the RNA oligonucleotides are mostly in the predicted conformations shown in Fig. 1C. These RNAs were labeled at their 5′-ends by addition of 32P-monophosphate and were incubated in the presence of RNase J1. Aliquots were removed at various times after addition of RNase J1 and were run on (a) an 8% denaturing polyacrylamide gel, to measure loss of label at the 5′-end; (b) a polyethyleneimine-cellulose sheet (thin layer chromatography), to measure release of labeled GMP by the 5′ to 3′-exonuclease activity; and (c) a 22.5% denaturing polyacrylamide gel, to measure accumulation of small fragments arising from endonucleolytic cleavage. Experiments were performed in quadruplicate. In Fig. 5, A–C, are shown graphs of the data from these assays on the three RNA substrates. Primary data for the assays of endonuclease activity on 22.5% denaturing polyacrylamide gels are shown in Fig. 5, D–F. Disappearance of full-length RNA occurred at similar rates for the three substrates (Fig. 5A), and these were inversely proportional to accumulation of labeled GMP (Fig. 5B). Thus, all three substrates were equally susceptible to the 5′ to 3′-exonuclease activity of RNase J1. However, the accumulation of small 5′-end-containing fragments, representing endonuclease activity, differed significantly among the three substrates. Quantitatively, there was a lower level of endonuclease fragments from the 3′-stem RNA than from the 3′-no-stem and the 3′-stem+5′-ext RNAs (Fig. 5C). Qualitatively, the 5′-end-containing fragments were of different sizes for the three substrates: for 3′-stem RNA (Fig. 5D), small fragments about 3–7 nt accumulated, with little accumulation of larger fragments; for 3′-no-stem RNA (Fig. 5E), fragments in a range from 3 to 20 nt were observed; for 3′-stem+5′-ext RNA, the pattern was shifted to mostly larger fragments (Fig. 5F). These observations could be explained in light of the structures of the three substrates. 3′-stem RNA has a 9-nt single-stranded region that precedes the stem structure. The data suggested that RNase J1 makes endonucleolytic cleavages in this single-stranded sequence but does not attack the stem structure. The small single-stranded target size and/or the proximity of the stem structure to the 5′-end in this substrate RNA reduces the rate of RNase J1 endonucleolytic cleavage relative to the other substrates (Fig. 5C). In the case of 3′-nostem RNA, RNase J1 can cleave at a wider range of phosphodiester bonds, as there is no inhibitory secondary structure present. For 3′-stem+5′-ext RNA, the single-stranded region preceding the stem structure is 33 nt long, and cleavage distal to the 5′-end is preferred. To analyze the effect of secondary structure on 5′ to 3′-processivity, the RNA oligonucleotide substrates were labeled at their 3′-ends by addition of [32P]pCp and were incubated in the presence of RNase J1. Note that the 3′-end-labeled substrates in these experiments had a 5′-hydroxyl end rather than the 5′-monophosphate end in previous experiments. Based on the published results describing 5′ to 3′-exonuclease activity of RNase J1 (5Mathy N. Benard L. Pellegrini O. Daou R. Wen T. Condon C. Cell. 2007; 129: 681-692Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), we expected that the activity of RNase J1 on 5′-hydroxylated RNA would be very similar to its activity on 5′-monophosphorylated RNA. The 3′-stem substrate showed an accumulation with time of a product that was several nucleotides shorter than the initial substrate (Fig. 6A). This product constituted 21.5% (average of four experiments) of the full-length RNA at time zero. From a sequencing gel, the size of the accumulated fragment was found to be 35–37 nt, with most of the product at 36 nt (Fig. 6B). Taking into account the added C residue at the 3′-end, this corresponded to a fragment whose 5′-end was 3 nt upstream of the base of the stem structure, thus suggesting that RNase J1 exonucleolytic processivity is hindered when it reaches within a few
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