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• W2600323898 abstract "Article31 March 2017free access Source DataTransparent process Rae1/YacP, a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis Magali Leroy Magali Leroy UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Jérémie Piton Jérémie Piton UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Laetitia Gilet Laetitia Gilet UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Olivier Pellegrini Olivier Pellegrini UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Caroline Proux Caroline Proux Transcriptome and EpiGenome, Biomics, Center for Innovation and Technological Research, Institut Pasteur, Paris, France Search for more papers by this author Jean-Yves Coppée Jean-Yves Coppée Transcriptome and EpiGenome, Biomics, Center for Innovation and Technological Research, Institut Pasteur, Paris, France Search for more papers by this author Sabine Figaro Sabine Figaro UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Ciarán Condon Corresponding Author Ciarán Condon [email protected] orcid.org/0000-0002-2199-9621 UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Magali Leroy Magali Leroy UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Jérémie Piton Jérémie Piton UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Laetitia Gilet Laetitia Gilet UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Olivier Pellegrini Olivier Pellegrini UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Caroline Proux Caroline Proux Transcriptome and EpiGenome, Biomics, Center for Innovation and Technological Research, Institut Pasteur, Paris, France Search for more papers by this author Jean-Yves Coppée Jean-Yves Coppée Transcriptome and EpiGenome, Biomics, Center for Innovation and Technological Research, Institut Pasteur, Paris, France Search for more papers by this author Sabine Figaro Sabine Figaro UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Ciarán Condon Corresponding Author Ciarán Condon [email protected] orcid.org/0000-0002-2199-9621 UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Author Information Magali Leroy1,‡, Jérémie Piton1,3,‡, Laetitia Gilet1, Olivier Pellegrini1, Caroline Proux2, Jean-Yves Coppée2, Sabine Figaro1 and Ciarán Condon *,1 1UMR 8261 (CNRS - Univ. Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris, France 2Transcriptome and EpiGenome, Biomics, Center for Innovation and Technological Research, Institut Pasteur, Paris, France 3Present address: Ecole Polytechnique Federale de Lausanne SV GHI UPCOL, Lausanne, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 58 41 51 23; E-mail: [email protected] The EMBO Journal (2017)36:1167-1181https://doi.org/10.15252/embj.201796540 See also: D Lalaouna & E Massé (May 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo. Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo. Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro. Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo. Synopsis A new bacterial endoribonuclease Rae1/YacP, founding member of the NYN subfamily of PIN domain proteins, cleaves mRNAs at specific codons through its association with the ribosome. The crystal structure of Bacillus subtilis RNase Rae1 reveals an N-terminal NYN domain that can be docked into the ribosome A-site. Potential substrates of Rae1 cleavage are revealed by RNA sequencing. Translation activity and correct reading frame are required for Rae1 cleavage of mRNA encoding a 17- amino acid peptide. Rae1 cleavage preferentially occurs immediately upstream of a lysine (AAG/AAA) codon. Introduction The Gram-positive model organism Bacillus subtilis currently has 19 known ribonucleases, consisting of eight exoribonucleases and 11 enzymes that cleave RNA endonucleolytically (Condon, 2014). Some of these have relatively specialised functions, such as RNase M5 in 5S rRNA maturation (Condon et al, 2001), and RNase P and RNase Z in tRNA maturation (Hartmann et al, 2009), while others have both stable RNA and mRNA substrates. One of the best known enzymes of this latter class is the 5′-3′ exoribonuclease RNase J1 that matures the 5′ end of 16S rRNA (Britton et al, 2007; Mathy et al, 2007) and is involved in the degradation of many mRNAs and mRNA fragments (Durand et al, 2012). RNase Y is also involved in both mRNA degradation and stable RNA processing, notably the scRNA (4.5S RNA) and the RNA subunit of RNase P (Lehnik-Habrink et al, 2011; Durand et al, 2012; Laalami et al, 2013; Gilet et al, 2015). Despite the large number of RNases identified in B. subtilis to date, there remain both orphan substrates, i.e. processed RNAs for which the enzymes are not yet known, and orphan enzymes, i.e. predicted RNases encoded by the genome of B. subtilis whose substrates have not been identified, that are ripe for further study. In this paper, we describe the characterisation of an orphan enzyme of B. subtilis called YacP that we have renamed Rae1 (ribosome-associated endonuclease 1). This enzyme was predicted to be a ribonuclease related to the PIN family (PilT N-terminal) of RNases (Anantharaman & Aravind, 2006). An extensive bioinformatic analysis showed that members of this so-called NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain enzymes are found in all three kingdoms of life and suggested that they are an ancient class of RNA processing enzymes that can be traced all the way back to the last universal common ancestor (LUCA). The rae1 gene in B. subtilis is the last cistron in a large operon encoding a number of proteins involved in translation, including the glutamyl- and cysteinyl-aminoacyl-tRNA synthetases (gltX and cysES), the 23S rRNA processing enzyme Mini-RNase III (mrnC) and the 23S rRNA methyltransferase (rlmB). This organisation is conserved in the Firmicutes (Fig EV1) and suggested that Rae1 may have a role in a translation-related process in these organisms. Click here to expand this figure. Figure EV1. Synteny of rae1 locus in bacteria Structure of rae1 locus in Firmicutes. The rae1 gene is shown in red. Genes are labelled for Bacillus subtilis. Conserved genes have the same colour. The per cent co-occurrence of genes immediately surrounding rae1 is given for 268 Firmicutes. Structure of rae1 locus in Cyanobacteria. The rae1 gene is shown in red. Genes are labelled for Synechocystis elongatus. Conserved genes have the same colour. The per cent co-occurrence of genes immediately surrounding rae1 is given for 35 Cyanobacteria. Download figure Download PowerPoint The NYN/PIN domain is found to be fused to a large variety of other domains throughout biology. Some of these are basic RNA binding domains, such as the Zn-finger CCCH domain, the K-homology (KH) domain, the RNA recognition motif (RRM) domain or pentatricopeptide repeats (PPR) (Anantharaman & Aravind, 2006). Much larger proteins related to the TetM/TetO family of tetracycline resistance proteins are also found with C-terminal NYN domains. Although the larger PIN family has been widely characterised (Arcus et al, 2011), only a few NYN subfamily proteins have been studied in any detail. MCPIP1/Zc3h12a/ Regnase-1 has been shown to be a Zn-finger containing RNase involved in dampening the mammalian immune response by cleaving the IL-6 and IL-12β mRNAs (Matsushita et al, 2009) and to play a role in antagonising micro-RNA (miRNA) biogenesis by cleaving pre-miRNAs in their terminal loops (Suzuki et al, 2011). It also has potent broad-spectrum antiviral activity through degradation of viral RNAs (Lin et al, 2013, 2014). The PPR-containing NYN protein PRORP is a protein-only form of the well-known tRNA 5′ processing ribozyme RNase P and is found in mammalian and plant organelles (Holzmann et al, 2008; Gutmann et al, 2012). The bacterial Rae1 enzyme consists of an N-terminal catalytic (PIN-like) domain and a short highly positively charged helical domain that resembles a number of ribosomal RNA binding proteins. Rae1 is conserved in the Firmicutes, the Cyanobacteria, algae and higher plants, where it is predicted to be localised principally to chloroplasts (Emanuelsson et al, 2007). Despite the identification of this putative novel B. subtilis ribonuclease as a founder member of the NYN/PIN family of RNases more than 10 years ago, nothing is yet known about its function in this organism. To get a handle on its biological role, we employed parallel approaches of crystal structure resolution and RNA sequencing (RNAseq) of a ∆rae1 mutant and a complemented strain. The crystal structure shows that Rae1 has an N-terminal NYN/PIN domain and a highly positively charged flexible C-terminal domain, comprised of two α-helices that likely serve in RNA binding. The RNAseq data show that Rae1 is involved in the degradation of a subset of B. subtilis mRNAs in rich medium. We further demonstrate that RNA cleavage by Rae1 is dependent on both the translation and reading frame of one of these substrates, making it a good candidate for a ribosome acceptor (A)-site ribonuclease. Results Crystal structure of Bacillus subtilis Rae1 (YacP) The wild-type (WT) Rae1 protein from B. subtilis and a fortuitously isolated mutant (W164L) were overexpressed in E. coli as C-terminal His-tagged fusion proteins. Both proteins behaved as monomers in solution with an apparent molecular weight of 25 kDa (see Appendix Fig S1A for the WT protein). The W164L derivative was first to crystallise and its structure was solved at 3.2 Å resolution using sodium iodide-soaked crystals and single isomorphous replacement with anomalous scattering (SIRAS) for phase determination. A different set of crystallisation conditions were subsequently obtained for the WT Rae1 protein and its structure was solved at 2.25 Å resolution by molecular replacement using the structure of the W164L mutant as a model. The refinement statistics for both proteins is given in Appendix Table S1. The N-terminal 120 amino acids of Rae1 form a PIN-related fold consisting of a central five-stranded parallel β-sheet associated with five helices, two behind and three in front (Fig 1A and Appendix Fig S1B). The first two helices of Rae1 (α1 and “α2”) are orthogonal to the plane of the β-sheet, while the remaining three are in antiparallel orientation. Figure 1. Structure of Rae1 monomer at 2.25 Å resolution Ribbon representation of Rae1 superimposed on a transparent surface map. α-helices and β-strands of the NYN/PIN domain are shown in red and blue, respectively. The C-terminal extension is shown in green. Helices α2 and α5 are not recognised as perfect α-helices and are thus labelled as “α2” and “α5”. Ribbon and stick representation of the Rae1 catalytic site superimposed on that of mammalian MCPIP1 (PDB 3V34). Key aspartic acid residues are shown as orange sticks. MCPIP1 bound to Mg2+ is shown in grey. Key residues of Rae1 are labelled in black; equivalent residues of MCPIP1 are labelled in grey. Surface potential map of Rae1, with positively charged residues in blue and negatively charged residues in red. Scale is from −5 to +5 kb T ec−1. Structure and charge distribution of r-protein S13 from E. coli, with positively charged residues in blue and negatively charged residues in red. Scale is from −5 to +5 kb T ec−1. Download figure Download PowerPoint The Rae1 catalytic site Unlike other PIN domain proteins in which the active site is partially covered by a “lid” (Appendix Fig S2), the active site of Rae1 is remarkably exposed at the surface of the protein (Fig 1A and B). This is an unusual configuration for a ribonuclease, leaving open the possibility that another partner binds to this surface of Rae1, creating a bipartite pocket around the active site. Three conserved aspartic acid residues, D7, D81 and D104, occupy positions equivalent to the magnesium (Mg)-coordinating Asp residues (D141, D226, D244) of MCPIP1 and are located at the C-terminal ends of strands β1 (D7), β4 (D104) and the N-terminal end of helix α4 (D81) (Fig 1B and Appendix Fig S1B and C). Mg2+ is not visible in the Rae1 structure, suggesting that it is provided by the RNA substrate. In MCPIP1, a fourth key Asp (D225) residue lies adjacent to D226 at the N-terminus of helix α4 (Xu et al 2012). D226 makes direct contact with the catalytic Mg2+, while D141, D225 and D244 bridge to the metal ion via a network of five water molecules. Interestingly, Rae1 differs from MCPIP1 and the other NYN proteins in the PDB by having only one Asp residue at the N-terminus of helix α4. A fourth Asp (D53), located at the C-terminus of β2, occupies a position not too distant from MCPIP1 D225 in the catalytic pocket and may serve a similar function (Fig 1B). In the current configuration, all four Asp residues of Rae1 are too far (>3.5 Å) from the predicted position of the Mg2+ ion to make direct contact. Asp7 and Asp53 are each hydrogen-bonded to a water molecule (water 116 in the A-subunit and 72 in the B-subunit, respectively) that could allow them to bridge to the Mg2+ ion (Fig EV2). Most likely the other Asp residues only move into place or bridge via additional water molecules once the substrate and Mg2+ are present, but it is clear that the network is incomplete without these two elements. Click here to expand this figure. Figure EV2. Hydrogen bond networks in the catalytic site of Rae1 crystal structure Water molecules in the catalytic site of the A-subunit of the Rae1 crystallographic dimer. Hydrogen bonds are shown as dotted yellow lines with distances are given in Å. Key catalytic residues are labelled. The Mg2+ ion (grey) is from MCPIP1 and was placed using the superposition shown in Fig 2. Water molecules in the catalytic site of the B-subunit of the Rae1 crystallographic dimer. Hydrogen bonds are shown as dotted yellow lines with distances are given in Å. Key catalytic residues are labelled. The Mg2+ ion (grey) is from MCPIP1 and was placed using the superposition shown in Fig 2. Download figure Download PowerPoint One of the distinguishing features of the NYN subfamily of PIN nucleases is a conserved Asn residue (Asn10), three residues downstream of the first catalytic aspartate (the DGYN motif) and a conserved small residue immediately (in this case a serine) upstream of the last catalytic aspartate (Asp104). The hydroxyl group of the Asn10 side chain hydrogen bonds with the backbone amino group of Ser103 to stabilise the relative positions of Asp7 and Asp104 (Figs 1B and EV2). Asn10 further stabilises the relative positions of these two key catalytic Asp residues via a network of water molecules (waters 2, 16 and 116 in the A-subunit). Putative RNA binding domain Crystals of the wild-type protein contain a dimer of Rae1 per asymmetric unit, with the dimer being formed through the intertwining of the C-terminal α-helices (Appendix Fig S1D). Since Rae1 is a monomer in solution, this dimer is not likely to be relevant in vivo. The C-terminal 50 amino acids of Rae1 form two highly positively charged α-helices (13 arginine or lysine residues) (Fig 1C), consistent with a role in RNA binding. Indeed, the C-terminal α-helical domain bears some overall charge and structural resemblance to a number of ribosomal proteins with positively charged helix-loop-helix motifs, most notably S13 (Fig 1D). In the monomeric conformation, the C-terminal domain of Rae1 is likely to be highly flexible and free to move to interact with RNA. Identification of potential targets of Rae1 To identify potential substrates of Rae1 in B. subtilis, we performed RNAseq analysis in triplicate on strains either lacking Rae1 (Δrae1) or complemented with a plasmid expressing a C-terminal Flag-tagged derivative of Rae1 (Rae1f) under control of an IPTG-dependent promoter. All annotated mRNAs, non-coding RNAs and RNA segments (5′/3′ UTRs) (Nicolas et al, 2012) were included in the analysis. Forty-six RNAs showed at least a 1.5-fold increased expression (P < 0.05) in the Δrae1 strain compared to WT, and we consider these to be potential direct targets of Rae1-mediated RNA degradation (Appendix Table S2). Thirteen RNAs had significantly lower levels than the WT strain and these likely represent indirect effects of the rae1 deletion or potential cases of RNA stabilisation following Rae1 cleavage. Interestingly, 17 of the 46 RNAs (37%) showing increased expression levels were members of the Fur (ferric uptake regulator) regulon, suggesting that the Δrae1 strain is subjected to a basal level of oxidative or iron (Fe3+) stress, despite the fact that it has no obvious growth phenotype. Eight of the up-regulated RNAs are members of the AbrB and/or SigW regulons. AbrB encodes a repressor involved in the regulation of starvation-induced processes (e.g. sporulation) in B. subtilis, while SigW is an extracellular function (ECF) sigma factor involved in the response to various types of envelope stress in B. subtilis. Globally, these results point to a potential role for Rae1 in stress management. Intriguingly, many of the candidate Rae1 targets (21 in total, both up- and down-regulated) encode proteins destined for the cytoplasmic membrane, periplasm or the cell wall. The vast majority of these cell envelope proteins are non-overlapping with the members of the Fur regulon. We also compared the global RNA expression patterns in the plasmid-complemented Δrae1 strain grown in the presence or absence of IPTG. Eleven RNAs showed decreased levels upon induction of Rae1 expression (Appendix Table S3). Seven of these showed the opposite effect in the rae1 deletion strain and we consider these to be among the best candidates for direct initiation of degradation by Rae1. Ten RNAs showed increased expression upon induction of Rae1 expression, but none of these were down-regulated in the Δrae1 strain. Validation of potential Rae1 targets by Northern blot We divided the RNAs into three categories for validation of the RNAseq results by Northern blot: (I) those showing effects restricted to the comparison between the WT and Δrae1 strains; (II) those showing effects only upon induction of Rae1 expression; and (III) those showing opposite effects in the two sets of strains. A Northern blot probed for the category I ykuNOP operon (Fur regulon) indeed showed increased expression in the rae1 deletion strain compared to WT and confirmed the lack of effect of Rae1 overexpression (Appendix Fig S3A). No effect of ∆rae1 was seen on the fur mRNA itself (Appendix Fig S3B), suggesting that the effect of Rae1 on members of the Fur regulon occurs through changes in Fur protein levels or in Fur activity. Northern analysis also confirmed the increased expression of the category II sigH mRNA only in the condition of Rae1 overexpression (Appendix Fig S3C), further validating the RNAseq data. Many of the seven candidates showing opposite effects in the Δrae1 and plasmid-complemented strain (category III) were encoded in the same operons. bmrC and bmrD are part of one operon encoding two subunits of an ABC multidrug exporter, previously known as YheIH (Torres et al, 2009; Galian et al, 2011). We confirmed the increased expression of this operon in the absence of Rae1 and its decrease in the plasmid-complemented strain by Northern blot (Appendix Fig S3D). The S1024, S1025, S1026 and yrzI (encoding a 49-amino acid peptide of unknown function) RNAs also form an operon that includes yet another RNA segment S1027 identified in an extensive study of the B. subtilis transcriptome (Nicolas et al, 2012; Fig 2A). Ribosome profiling data suggest that a number of these short RNA segments are translated in rich medium, notably S1027, S1025 and to a lesser extent S1024, encoding potential peptides of 38, 17 or 52 amino acids, respectively (Li et al, 2012). For the rest of this study, we will focus on the effect of Rae1 on expression of the yrzI operon. Figure 2. Role of Rae1 in yrzI mRNA turnover Structure of the yrzI operon. Putative promoters are represented by rightward arrows and putative transcription terminators by hairpin structures. Transcripts from this locus that include yrzI are shown as wavy lines. Northern blot showing expression of the yrzI transcript in WT, ∆rae1 and plasmid-complemented strains grown in the absence (−) and presence (+) of IPTG for 1 h. Number of biological replicates (n) = 3. The blot was probed with an oligonucleotide complementary to yrzI (CC1589) indicated by the black bar in panel (A). The correspondence between bands and transcripts shown in panel (A) is given to the right of the blot. R-T4 refers to the 521-nt species extending from 21 nt upstream of the yrzI coding sequence to the main operon terminator (T4). A Western blot showing the overexpression of Rae1 in the presence of IPTG is shown underneath the Northern blot; note that Rae1 is undetectable in WT cell extracts, even upon overexposure (n = 2). Northern blot showing levels of yrzI mRNA at times after rifampicin addition (n = 2). Northern blot showing that Rae1 catalytic activity is required for accumulation of yrzI mRNAs. Plasmid pDG-Rae13fMut expresses the 3×Flag-tagged (3f) catalytic mutant D7N D81N (n = 2). Data information: Blots in panels (B–D) were rehybridised with a probe complementary to 16S rRNA (oligo CC058) as a loading control. Molecular weight markers (kb) are shown to the left of autoradiograms. Source data are available online for this figure. Source Data for Figure 2 [embj201796540-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint The rae1 deletion increases the stability of the yrzI operon mRNAs Deletion of the rae1 gene led to an accumulation of three yrzI-containing transcripts of ~2.4, ~0.8 and ~0.5 kb in size (Fig 2B, lane 2). The two larger transcripts likely correspond to primary transcripts from the P1 and P2 promoters upstream of the yrhF and yrhG genes, respectively, while the 5′ end of the ~0.5-kb transcript maps to 21 nt upstream of the yrzI coding sequence (Fig 3A, lane 2), with no obvious corresponding promoter. Induction of plasmid-borne Rae1 expression reduced the level of these transcripts significantly (Fig 2B, lane 4), although not quite to WT levels, suggesting that the Flag tag may interfere slightly with Rae1 activity. Figure 3. Mapping of Rae1 cleavage site in the yrzI mRNA Primer extension assay (oligo CC1671) on total RNA isolated from wild-type and strains lacking Rae1 (∆rae1), RNase J1 (∆rnjA) and both (∆rae1 rnjA) (n = 2). Sequence lanes are labelled as their reverse complement to facilitate direct read-out. The left and right panels represent different exposures of the same gel. Reverse transcriptase (RT) stops corresponding to the T2 and T3 terminator structures, the predicted P3 promoter and a series of 5′ ends mapping within the yrzI ORF in the ∆rae1 rnjA strain are indicated. Mapping of the Rae1 cleavage site to the secondary structure of the yrzI-S1024 region predicted by LocaRNA (Smith et al, 2010) using sequences shown in panel (C). Coordinates are given relative to the AUG start codon of S1024. The start and stop codon of the putative 17-aa S1025 ORF are also boxed. Conservation of S1025 nucleotide and amino acid sequence in Bacilli. Conserved nucleotides are marked with an asterisk. Conserved amino acids at the C-terminus of the peptide are indicated. The SD sequence is underlined in red and the start codon highlighted in green. The Rae1 cleavage site in Bacillus subtilis is indicated with a vertical red arrow. Source data are available online for this figure. Source Data for Figure 3 [embj201796540-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint We confirmed that the effect of the rae1 deletion on yrzI transcript levels occurred at the level of mRNA stability. In the WT strain, two of the three yrzI transcripts were only barely detectable at the zero time point and all three disappeared rapidly after rifampicin addition (Fig 2C). All three transcripts were clearly stabilised in the Δrae1 strain but to varying degrees. The ~0.5-kb RNA showed little if any degradation over the time course of the rifampicin experiment, showing that the degradation of this short yrzI transcript is strongly dependent on Rae1. We performed an additional experiment to confirm that the levels of the yrzI transcripts observed in vivo were dependent on the catalytic activity of Rae1. The crystal structure of Rae1 allowed us to identify the key amino acids likely to be involved in Mg binding (D7, D81 and D104). As a tool to allow us to better co-immunoprecipitate RNAs bound to Rae1 (to be reported elsewhere), we constructed a second Rae1 complementation strain expressing either a WT or catalytic mutant (D7N, D81N) of Rae1 bearing a 3×Flag tag (Rae13f). An equivalent mutation in one of these residues (D141N) was shown to functionally inactivate MCPIP1 (Matsushita et al, 2009). Induction of the expression of the 3×Flag derivative of WT Rae1 complemented the rae1 deletion strain to the same extent as the 1×Flag construct in terms of the yrzI expression profile seen in Northern blots (Fig 2D; compare lanes 4 and 5). However, the Rae13f D7N D81N mutant construct failed to complement upon induction of expression (Fig 2D; lane 6), confirming that Rae1 catalytic activity is required for degradation of yrzI mRNAs. Mapping of the putative Rae1 cleavage site Our data suggested that the primary pathway to initiate degradation of the ~0.5-kb yrzI mRNA is through cleavage by Rae1. We reasoned that mapping of the Rae1 cleavage site would require stabilisation of the downstream cleavage product by inactivating the 5′–3′ exoribonuclease RNase J1. We therefore performed primer extension assays on total RNA isolated from WT, Δrae1, ΔrnjA and Δrae1 rnjA double mutant strains, using an oligonucleotide that hybridised to the coding sequence of S1024. In the WT strain, we observed a weak band corresponding to a 5′ end within the putative S1025 ORF, in the loop of a predicted stem-loop structure in untranslated forms of the RNA (Fig 3A and B). In the ΔrnjA strain, this 5′ end was significantly more abundant, confirming that RNase J1 indeed degrades the downstream product of Rae1 cleavage. In the double Δrae1 rnjA mutant, this 5′ end was absent as expected and instead a number of new 5′ ends mapping to within the upstream yrzI ORF were visible. These data suggest that another (unknown) enzyme can give RNase J1 access to the yrzI mRNA in the absence of cleavage by Rae1. However, the strong stabilisation of the ~0.5-kb yrzI-S1024 seen in the Δrae1 strain indicates that this alternative pathway is not very efficient. Addition of a Rae1 cleavage site destabilises a heterologous mRNA The Rae1 cleavage site mapped to the yrzI-S1024 intergenic region, within the putative 17-aa ORF encoded by S1025 (Fig 3C). We asked whether insertion of this 156-nt sequence into the 3′UTR of the highly stable hbs mRNA could destabilise it in a Rae1-dependent manner. The hbs gene encodes an orthologue of the E. coli HU protein and is essential in B. subtilis. We therefore used an ectopic chromosomally expressed deletion derivative of this mRNA called hbs∆ that we have previously characterised extensively (Fig 4A) (Daou-Chabo & Condon, 2009; Daou-Chabo et al, 2009). Like the native hbs mRNA, the hbs∆ construct yields three transcripts corresponding to expression from promoters P3 (P3-ter) and P1 (P1-ter) and a highly stable ribosome-protected spe" @default.
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• W2600323898 date "2017-03-31" @default.
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• W2600323898 title "Rae1/YacP, a new endoribonuclease involved in ribosome‐dependent mRNA decay in <i>Bacillus subtilis</i>" @default.
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