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Ymdb: a Stress-Responsive Ribonuclease-Binding Regulator of E Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity Kwang-sun Kim, Robert Manasherob, and Stanley N. Cohen1 Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA The broad cellular actions of RNase III family enzymes include ribosomal RNA (rRNA) processing, mRNA decay, and the generation of noncoding microRNAs in both prokaryotes and eukaryotes. Here we report that YmdB, an evolutionarily conserved 18.8-kDa protein of Escherichia coli of previously unknown function, is a regulator of RNase III cleavages. We show that YmdB functions by interacting with a site in the RNase III catalytic region, that expression of YmdB is transcriptionally activated by both cold-shock stress and the entry of cells into stationary phase, and that this activation requires the ␴-factor-encoding gene, rpoS. We discovered that down-regulation of RNase III activity occurs during both stresses and is dependent on YmdB production during cold shock; in contrast, stationary-phase regulation was unperturbed in ymdB-null mutant bacteria, indicating the existence of additional, YmdB-independent, factors that dynamically regulate RNase III actions during normal cell growth. Our results reveal the previously unsuspected role of ribonuclease-binding proteins in the regulation of RNase III activity. [Keywords: Ribonuclease III; RpoS; cold shock; RNA decay; RNA processing] Supplemental material is available at http://www.genesdev.org. Received August 15, 2008; revised version accepted October 16, 2008. RNase III family ribonucleases, which cleave dsRNA to protein-coding RNAs (Murchison and Hannon 2004), yield 5Ј phosphate and 3Ј hydroxyl termini, are remark- and mRNAs that encode the exoribonuclease PNPase able for both the extent of their evolutionary conserva- (Pnp) (Régnier and Portier 1986), Rnc itself (Matsunaga et tion in prokaryotic and eukaryotic cells and the diversity al. 1996a,b), or phage and plasmid proteins (Nicholson of their biological roles (Court 1993; Nicholson 2003; 1996, 2003). However, notwithstanding the multiplicity Drider and Condon 2004; MacRae and Doudna 2007). and breadth of functions of RNase III, rnc-null mutant E. Containing 226 amino acids, the 25.6-kDa peptide en- coli cells are viable and show no phenotypic abnormali- coded by the rnc gene of Escherichia coli (EC 3.1.26.3) is ties except for a slightly impeded rate of growth structurally the least complex and functionally perhaps (Babitzke et al. 1993) and minimally defective transla- the most extensively studied RNase III family member. tion of certain mRNAs (Talkad et al. 1978). Rnc monomers, which include an N-terminal catalytic The first suggestion that the ribonucleolytic actions of domain and a C-terminal dsRNA-binding domain RNase III may be regulated came from the observation (dsRBD) (Nashimoto and Uchida 1985), dimerize to form by Makarov and Apirion in 1992 that extracts of E. coli the enzymatically active protein (Dunn 1982; Nicholson cells contain an ∼17-kDa protein that can inhibit in vitro 2003). RNase III-dependent processing of p10Sa RNA (Makarov E. coli RNase III was first identified by its ability to and Apirion 1992)—a 10Sa RNA precursor (also known catalyze cleavage of ribosomal RNA (rRNA) precursors as ssrA RNA and tmRNA) that has an important role in during ribosome biogenesis (for review, see Dunn 1982). the recycling of ribosomes from defective mRNAs (Rich- Subsequently, this enzyme has been shown to mediate ards et al. 2008). However, the nature of this RNase III- the maturation and/or degradation of a variety of tran- inhibiting moiety and its possible biological role(s) has scripts, including tRNA precursors (Régnier and Grun- remained obscure for almost two decades. Using a func- berg-Manago 1989), conditionally expressed small non- tion-based screen to identify genes encoding proteins that regulate endonucleolytic cleavages by RNase III in vivo, we discovered a highly conserved bacterial protein YmdB that inhibits RNase III actions by binding to the 1Corresponding author. region required for dimerization/activation of the en- E-MAIL [email protected]; FAX (650) 725-1536. zyme. We show that production of YmdB and the con- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1729508. Freely available online through the Genes & Development Open Access sequent inhibition of RNase III function are modulated option. in response to cellular and environmental stresses. Our GENES & DEVELOPMENT 22:3497–3508 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 3497 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Kim et al. results reveal a novel mechanism for the dynamic regu- cifically from inhibition of Rnc activity was confirmed lation of ribonuclease activity by alterations in cell using PL-putL-lac fusion transcripts, which are attacked physiology. at a site immediately 3Ј of putL to generate an 80-nucleo- tide (nt) product (Sloan et al. 2007); turn on of the YmdB Results expression led to accumulation of the 140-nt unproc- essed transcript and concurrent reduction of the 80-nt Screening of RNase III regulators in E. coli RNase III cleavage product (Fig. 1C). To identify trans-acting regulators of RNase III activity, Inhibition of RNase III cleavages by purified His-tagged we introduced a library of plasmid-borne E. coli genes YmdB protein was demonstrated biochemically in vitro [the ASKA library, which includes all known E. coli and the inhibition was shown to be dependent quantita- ORFs expressed from an IPTG-inducible promoter tively on YmdB using R1.1 RNA (60 nt in size), a small (Kitagawa et al. 2005), into an E. coli strain (RS7305) RNase III substrate containing a sequence that is the site containing a single copy of a rncЈ-ЈlacZ reporter gene of RNase III-mediated processing of the phage T7 poly- fusion (Matsunaga et al. 1996a)]. Earlier work has shown cistronic early mRNA precursor (Fig. 1D; Amarasinghe that RNase III cleaves its own transcript, and conse- et al. 2001). The distinctly different YmdB/RNase III ra- quently that ␤-galactosidase production from this fusion tios found to affect RNase III activity in vivo and in vitro construct is increased approximately ninefold in cells imply that experimental conditions employed for the in containing a missense mutation that decreases RNase III vitro assays do not entirely mimic the physiological activity (Matsunaga et al. 1996a,b). We reasoned that state. cleavage of the rncЈ-ЈlacZ transcript would also be af- fected by down-regulators of Rnc activity encoded by YmdB interaction with RNase III ASKA library ORFs. Regulation of expression of ␤-galac- tosidase from the rncЈ-ЈlacZ fusion was monitored col- Notwithstanding the ability of YmdB to inhibit RNase orimetrically on MacConkey-Lac and S-gal plates as de- III-mediated cleavage of R1.1 RNA, gel shift analysis scribed in the Materials and Methods. Two colonies that failed to detect binding of YmdB to this substrate. In- showed reproducibly increased color intensity on culture stead, multiple lines of evidence indicated that YmdB plates containing IPTG were obtained and the phenotype interacts with the RNase III protein. This interaction was confirmed. Sequence analysis of ASKA inserts in was suggested initially by our detection of an RNase III- ∼ plasmids isolated from these colonies identified ymdB sized 26 kDa protein, using rabbit polyclonal antibody (b1045), which is located at the 23.82-min position of the generated against E. coli RNase III, in histidine-tagged 2+ E. coli chromosome and encodes a putative 177-amino- YmdB preparations purified by Ni column chromatog- + acid 18.88-kDa protein of previously unknown function raphy from rnc —but not from rnc mutant—bacteria (Swiss-Prot P0A8D6). Swiss-Prot database analysis of the (data not shown). More direct evidence of the ability of putative YmdB protein indicates that amino acids 1–175 YmdB and RNase III to interact was obtained in experi- comprise a macro (A1pp) domain characteristic of Appr- ments showing that biotin-tagged YmdB preparations 1Љ-p processing family proteins, which catalyze the con- purified by a streptavidin bead-binding procedure (see version of ADP-ribose-1Љ-monophosphate (Appr-1Љ-p) to Materials and Methods) also included an RNase III-size ADP-ribose in eukaryotes (Culver et al. 1994). protein detected on Western blots by RNase III antibody (Fig. 2A). The observed copurification of this protein with biotin-tagged YmdB was unaffected by treatment of Effect of YmdB on cleavage of RNase III targeted YmdB with ribonuclease A (+ vs. −), implying that inter- transcripts action of this protein with RNase III does not require an Addition of IPTG to cultures of bacteria expressing the RNA intermediate. Independently of these results, high- ASKA library-derived plasmid-borne ymdB insert under throughput MALDI-TOF screening (Butland et al. 2005) control of the plac promoter increased production of a of protein complexes isolated from E. coli cells has iden- protein migrating in polyacrylamide gels at the position tified YmdB in protein complexes containing RNase III expected for YmdB (Fig. 1A, left). This increase was as- (A. Emili, pers. comm.). sociated with reduced cleavage of transcripts containing Physical interaction between YmdB and RNase III in RNase III targeted sites in rnc and pnp mRNAs fused to vivo was further confirmed and the RNase III segment lacZ (Matsunaga et al. 1996a; Beran and Simons 2001) as required for the interaction was identified by analysis, in evaluated quantitatively by measuring ␤-galactosidase cells mutated in the chromosomal rnc gene, of truncated activity encoded by lacZ (Fig. 1A, middle). Using purified RNase III proteins expressed from the pKSC1-Rnc plas- RNase III and YmdB to quantify the cellular levels of mids and captured by YmdB attached to streptavidin- these proteins in vivo by Western blotting, we determined coated beads (Fig.
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