The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration

Abstract

Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.

Figure 1.

Cellular abundance of different types of Salmonella Hfq-dependent sRNAs. Expression of Hfq-bound sRNAs in mid-exponential (top) or early stationary phase (bottom) in LB-broth. The list of Hfq-bound sRNAs was generated from Hfq-CoIP and Hfq-CLIP data (17,44). Expression values calculated as transcripts per million collected from the Salcom database (Supplementary Table S4) (33). The RNase E processed 3′ UTR candidates were predicted with TIER-seq data (28) and are marked in red. Experimentally confirmed copy numbers of sRNAs (17,45,46) are given above.

Figure 2.

NarS is a 3′ UTR derived sRNA from nitrate transporter gene narK. (A) Genomic context of narK and NarS. Alignment of narK 3′UTRs of selected enterobacterial genera is shown below. Conserved nucleotides are marked in red. The stop codon of the narK ORF is boxed. The Rho-independent terminator is underlined and potential seed-sequence is highlighted. ‘::’ near the stop codon in the E. coli sequences (Escherichia) represents a 170-nt insertion. (B) Expression of NarS in Salmonella under anaerobic shock. Salmonella SL1344 cells were grown to OD₆₀₀ of 0.3 in LB broth, then filled into 50 ml closed Falcon tube and incubated without agitation at 37°C for 2 h. Total RNA was isolated at indicated time points post treatment and analyzed on a northern blot. 5S RNA was probed as a loading control. (C) Predicted secondary structure of NarS-S. Hfq-binding regions identified by UV CLIP-seq analysis are marked with red letters. UV-crosslinking induced mutations are indicated by asterisks. The potential seed sequence is highlighted in yellow.

Figure 3.

NarS is an Hfq-dependent sRNA processed by RNase E. (A) Expression of NarS requires the transcriptional regulators NarL and FNR, and the RBP Hfq. Total RNA was isolated from the indicated strains and analyzed by northern blotting. (B) Expression of NarS in Salmonella lacking different narK regions. ΔnarKΔNarS: deletion from narK promoter to terminator; ΔP_narK, deletion of the narK promoter region and retained the whole ORF; ΔP_narkΔnarK: deletion from narK promoter to 100 nt upstream of NarS-S 5′ end; ΔnarK: deletion from narK transcriptional start site to 100 nt upstream of NarS-S 5′ end, while retained the promoter of narK. NarS was only detected in this strain. (C) Expression of NarS from pZE12-based plasmids carrying different narK genomic regions. P_narK-narK-NarS: plasmid carrying native narK promoter and the whole narK transcript include the 3′ UTR; P_narK-narK: plasmid carrying native narK promoter and narK ORF without the 3′ UTR; 100 bp + NarS: plasmid carrying NarS-S plus upstream 100 bp regions. P_narK-narK-NarS^UUC-CAG: same as P_narK-narK-NarS but with mutations around NarS-S 5′ end, to mutate the RNase E-recognition motif. (D) Northern blot analysis of NarS expression in the RNase E temperature sensitive strain rne-3071 (rne-TS) and the wild-type allele (rne-ctr). Bacterial cells were subjected to anaerobic shock after aerobic growth to an OD₆₀₀ of 0.3. ‘–’ indicates RNA samples before anaerobic shock and temperature shift.

Figure 4.

NarS represses expression of the nitrite transporter NirC. (A) RNA-seq based comparison of genome-wide mRNA expression (FPKM values) between NarS deleted (ΔnarKΔNarS + pJV300) and overexpression (ΔnarKΔNarS + pP_L-NarS) strains. Bacteria were subjected to anaerobic shock for 30 min at OD₆₀₀ of 0.3. (B) Western and northern blot analyses of NirC::3xFLAG protein and NarS RNA levels, respectively, in different mutant strains. Protein and RNA samples were collected at 0 and 30 min after anaerobic shock at OD₆₀₀ of 0.3. GroEL and 5S rRNA were loading controls. Relative fold changes to wild-type strain are marked above. (C) Regulation of the NirC::FLAG protein by ectopically expressed NarS from high-copy plasmids that carry different regions of nark-NarS locus. Only strains with a wild-type NarS seed sequence showed reduced NirC expression (lane 3, P_nark-narK-NarS and line 6, P_L-NarS). Relative fold changes to wild-type strain are marked above. (D) Colony fluorescence of ΔnarKΔNarS strain carrying either control plasmid pXG-1 (upper) or pXG30-sfGFP in-frame fused with nirD-nirC intergenic region (lower), combined with pZE12 based plasmids expressing empty, wild type or mutant NarS. Fluorescence was quantified by FACS and normalized to strain with pJV300 plasmid as shown in (E), data represent three independent experiments (mean ± SD). (F) Interaction between NarS and nirC 5′ UTR mRNA predicated by IntaRNA (upper panel) and conservation of nirC Shine-Dalgarno region among of selected enterobacterial genera (lower panel). Conserved nucleotides are marked in red. The base pairing region is highlighted in yellow. The nirC start codon is boxed.

Figure 5.

Extracellular nitrite concentrations in anaerobic growth media. Bacterial cells were grown in M9CA medium to OD600 of 0.3 and then supplemented with sodium nitrate (final concentration at 20 mM). Cultures were transferred into 15 ml closed Falcon tubes and incubated without agitation. Samples were collected at 0, 15, 30, 45 and 60 min after anaerobic shock. Supernatants were analyzed to determine nitrite concentration using a colorimetric assay as described in materials and methods.

Figure 6.

NarS selectively regulates the expression of nirC mRNA within the nirBDC-cysG operon. (A) Genomic organization of the nirBDC-cysG operon. Northern blot detection of mRNA isoforms and their predicted length are shown below. ‘PPP’ refers to a tri-phosphorylated 5′ end and ‘P’ refers to a mono-phosphorylated 5′ end, as determined by 5′ RACE. (B) Analyses of NirB::FLAG, NirD::FLAG and CysG::FLAG proteins and NarS RNA levels in individually Flag-tagged strains. Wild-type or mutant NarS were constitutively expressed from high-copy plasmids (pP_L-NarS). Relative fold changes to wild-type strain are marked above. (C, D) Northern blot analyses of nirBDC-cysG mRNA expression. Total RNA was collected from anaerobic shock treated cells and separated by 1.2% agrose gel. λ-EcoT14 I/Bgl II digest was loaded as marker and rRNA was visualized by EtBr staining before transferring to membrane. Multiple bands were detected by the anti-cysG (C) and anti-nirB (D) probes, as marked by arrows. The reduced levels of nirC-cysG in ΔnarK was due to altered NarS expression in this strain.

Figure 7.

An intergenic terminator is required for selective regulation by NarS. (A) Genomic context of the putative terminator downstream of nirD. Arrows refer to the 5′ ends of nirC mRNA as detected by 5′ RACE. (B, C) Expression of nirBDC-cysG and different isoforms in the RNase E temperature sensitive strain. Bacteria were grown to OD₆₀₀ of 0.3, filled into 50 ml closed Falcon tubes and incubated without agitation at 28°C or 44°C for 30 min. ‘–’ indicates RNA samples before anaerobic shock and temperature shift. Total RNA was separated on a 1.2% agarose gel for northern blotting analysis. λ-EcoT14 I/Bgl II digest was loaded as marker and rRNA was visualized by ethidium bromide staining before transfer to membrane. (D, E) Expression of nirBDC-cysG and different isoforms in nirD terminator and U-track deleted strain (ΔsU) in the presence and absence of NarS. (F, G) Fluorescence of ΔnarKΔNarS strain carrying either control plasmid pXG-1 (control), or pXG30-sfGFP in-frame fused with wild-type (nirDC-WT) or ΔsU (nirDC-ΔsU) nirD-nirC intergenic region combined with empty vector pJV300 (upper half) or NarS expressing plasmid (pP_L-NarS) (lower half). Fluorescence was quantified in a strain carrying pZE12 empty vector (upper half) or pZE12 expressing NarS (upper half). Fluorescence was normalized to a strain with control plasmid pJV300. Data represent three independent experiments (mean ± SD).

Figure 8.

Coordinated regulation of nitrate metabolism by NarK and the 3′ UTR sRNA NarS. Under anaerobic conditions with abundant nitrate, transcriptional regulators FNR and NarL activate the transcription of nitrate/nitrite antiporter NarK, respiratory nitrate reductase narGHI and operon nirBDC-cysG. Nitrate is imported by NarK into the bacterial cytoplasm where it is reduced to nitrite by the respiratory nitrate reductase NarG. To protect the cytoplasm from excess nitrite toxicity, reductase NirBD catalyzes subsequent reduction of nitrite to ammonia. NarS is processed from the narK mRNA by RNase E to repress the expression of nitrite transporter NirC to limit nitrite import.