An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs

Abstract

The small RNAs associated with the protein Hfq constitute one of the largest classes of post-transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free-standing genes. Here, deep sequencing of Hfq-bound transcripts from multiple stages of growth of Salmonella typhimurium revealed a plethora of new small RNA species from within mRNA loci, including DapZ, which overlaps with the 3' region of the biosynthetic gene, dapB. Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and is controlled by HilD, the master regulator of Salmonella invasion genes. DapZ carries a short G/U-rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB-like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq-dependent trans-acting RNA. Our atlas of Hfq targets suggests that the 3' regions of mRNA genes constitute a rich reservoir that provides the Hfq network with new regulatory small RNAs.

Figure 1

Dynamic sRNA profiles of Hfq over bacterial growth. (A) Growth curve of Salmonella grown for 14 h in LB at 37°C, 220 r.p.m. Time points when culture samples were withdrawn for Hfq co-immunoprecipitation are indicated. (B) Distribution of reads matching experimentally validated sRNAs in Hfq-coIP cDNA libraries at several stages of growth. Percentage indicates the reads of a given sRNA compared to all sRNAs in a cDNA library. The relative amount of reads and enrichment factors for individual sRNAs are listed in Supplementary Table S3. ON: overnight culture.

Figure 2

Expression analysis of 3′ UTR-derived sRNAs. Total RNA was prepared from wild-type Salmonella grown in LB at the time points indicated in Figure 1A, and subjected to northern blot analysis. (*) denotes detection of associated full-length mRNAs in the cases of DapZ, STnc850, STnc870 and STnc2090. The position of sRNAs, the name and the length of flanking genes, as well as the length (bp) of the intergenic regions are shown in the schematic presentations below the blots. All sRNAs identified are in close proximity to, or even partially overlap with upstream genes. Hybridization probes are listed in Supplementary Table S8.

Figure 3

Promoter and sequence analysis of DapZ sRNA. (A) Sequence alignment of the dapB 3′ coding sequence (CDS, grey box) and 3′ UTR of related enterobacterial species. The Salmonella dapZ sequence and its homologous sequences in other species are shown in bold. The arrow denotes the +1 site of dapZ in Salmonella. Putative promoter motifs within the dapB CDS and the ρ-independent terminator sequence are indicated. The conserved GU-rich motif R1 (boxed) is found in most species but absent in E. coli and Shigella. ST: Salmonella typhimurium; SB: S. bongori; ET: Enterobacter spp.; CN: Cronobacter spp.; SE; Serratia spp.; PA: Pantoea spp.; KP: Klebsiella pneumonia; YP: Yersinia pestis; ER: Erwinia spp.; CR: Citrobacter rodentium; SF: Shigella flexneri; EC: E. coli; Con: consensus sequence. Below left: DapZ GU-rich motif R1 (boxed) displays high similarity to GcvB R1. Below right: The secondary structure of DapZ sRNA (the GU-rich motif R1 is boxed) predicted by Mfold and validated by structure probing (Supplementary Figure S11). (B) The DNA sequence downstream of the dapB start codon down to the ρ-independent terminator was cloned into a high-copy plasmid, and the expression of DapZ in wild-type Salmonella at OD₆₀₀ of 2 was determined by northern blot. (*) denotes read-through to the rrnB terminator encoded on the plasmid. (C) Identification of the primary transcription start site of dapZ by 5′ RACE. PCR products were analysed on a 4% agarose gel. The arrow indicates the band corresponding to the primary transcript, which is enriched by RNA pre-treatment with TAP. The appearance of a weaker RACE signal for the full-length DapZ sRNA in the ‘− TAP’ lane may be attributed to the activity of RppH or a related pyrophosphohydrolases (Deana et al, 2008), which converts to 5′PPP to 5′P ends in vivo. The shorter RACE product in the TAP− lane represents a processing intermediate at nucleotide U12 of DapZ (unpublished results), and as expected, is not found in the TAP+ reaction. (D) Schematic drawing showing that the DapZ sRNA is transcribed from the 3′ UTR of the dapB gene and enriched at early stationary phase in the Hfq coIP library.

Figure 4

DapZ is activated by the Salmonella-specific virulence regulator HilD. (A) Salmonella wild-type as well as several deletion mutant strains were grown in LB to an OD₆₀₀ of 2, and total RNA was probed for DapZ expression by northern blot. 5S rRNA served as loading control. (B) Salmonella wild-type, ΔhilD and ΔSPI-1 strains were transformed with an arabinose-inducible pBAD control plasmid, pBAD-hilD or pBAD-hilA (ΔSPI-1 strain) and cultivated in LB to OD₆₀₀ of 1.0. Expression from pBAD plasmids was induced by addition of 0.2% L-arabinose (final conc.) for ∼45 min and DapZ levels were determined by northern blot. (C) Western blot analysis of GFP reporters in which dapZ homologues from several related enterobacteria were fused to a promoterless gfp gene: Salmonella typhimurium (ST), Yersinia pestis (YP), Klebsiella pneumonia (KP), Citrobacter rodentium (CR) and E. coli (EC). P_ompC and P_hilA are the control promoter regions known to be non-responsive or responsive, respectively, to HilD. E. coli co-transformed with the indicated GFP-reporter plasmids as well as the pBAD-hilD (hilD) plasmid were grown in LB for 2 h in presence of 0.0004% L-arabinose after reaching OD₆₀₀ of 0.5. Probing for GroEL served as loading control.

Figure 5

DapZ is a repressor of the opp and dpp operons. (A) Microarray analysis of genes affected by pulse expression of DapZ compared to pBAD control vector in Salmonella. Salmonella dapZ mutants containing a pBAD control vector or pBAD-DapZ plasmid were grown in LB until OD₆₀₀ of 1.5 and then 0.2% L-arabinose was added to both cultures for 10 min to induce DapZ expression (Supplementary Figure S12). Global transcriptome changes were scored on Salmonella-specific microarrays. Genes which show ⩾2-fold change (P-value <0.1) are marked in red. (B) Heat map analysis of microarray results of genes regulated by pulse expression of wild-type DapZ and DapZ-ΔR1. All the experimentally validated direct targets of the Salmonella GcvB sRNA (Sharma et al, 2007, 2011) are shown, and the fold-change values are listed in Supplementary Table S5. (C) Endogenous DapZ represses oppA and dppA protein synthesis in Salmonella at OD₆₀₀ of 2. Translational lacZ fusions were constructed in the Salmonella chromosome by fusing lacZ to the 17th codon of oppA or the 10th codon of dppA. β-Galactosidase activity in wild-type, ΔdapZ, ΔgcvB and ΔdapZΔgcvB double mutant Salmonella was determined in triplicates.

Figure 6

DapZ targets C/A-rich sites in oppA and dppA. Identification of duplex formation sites by in vitro secondary structure probing using 5′ end-labelled DapZ sRNA (A), oppA (B) and dppA (C) mRNA leaders. Radio-labelled RNA (∼5 nM) was subjected to RNase T1 or lead (II) cleavage in absence or presence of unlabelled 100 nM (+), 500 nM (++) oppA and 20 nM (+), 100 nM (++) dppA in (A), 100 nM (+), 500 nM (++) DapZ in (B), and 20 nM (+), 100 nM (++) DapZ in (C). C: control RNA, T1: RNase T1 ladder, OH: alkaline ladder. The G residues are labelled relative to the translation start site in oppA and dppA mRNA leaders. The regions protected by duplex formation with cold RNA are marked with red square brackets. The DapZ R1 region is indicated with a blue bar. (*) denotes a structure rearrangement in dppA, which we tentatively exclude as another targeting site, because the GAGUAUUUCCUU nucleotides (+3 to +14 of dppA) in question have no obvious complementarity with DapZ. Thus, there might be further structural rearrangement of the mRNA upon DapZ binding, which would also explain the difference in migration of dppA leader RNA in native gels (Supplementary Figure S8). (D) Validation of the base-pair interactions using translational oppA::gfp and dppA::gfp reporter gene fusions by compensatory base-pair exchange in vivo. Salmonella strains containing both a gfp reporter plasmid and a vector overexpressing DapZ were grown overnight in LB and analysed by flow cytometry. Overexpression of a ∼50 nt nonsense RNA was used as control (pJV300). (E) RNA duplexes formed between DapZ sRNA and the dppA or oppA leaders. Nucleotides in bold in oppA and dppA were previously shown to be involved in binding to GcvB sRNA (Sharma et al, 2007); the GcvB-dppA and GcvB-oppA interactions are shown for comparison below. Point mutations introduced for compensatory base-pair exchange experiments are indicated. The ribosome binding site and the start codon are marked in orange.

Figure 7

Biogenesis of sRNAs from bacterial UTRs. A 3′ UTR-derived small RNA can be either transcribed from its own promoter in the upstream coding sequence, or generated by internal processing of the associated mRNA. The common denominator is the shared use of the ρ-independent terminator of the mRNA. Hfq plays a seminal role in either pathway such that it facilitates the base pairing of the 3′ UTR-derived sRNA with trans-encoded target mRNA(s), but it may also participate in recruiting a nuclease (such as RNase E) to the 3′ end of the mRNA in the case of processing.