ABSTRACT
Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5’UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa.
IMPORTANCE The ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.
INTRODUCTION
The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen infecting humans that thrives in diverse environments. It can cause serious biofilm-associated infections in burn wounds, indwelling devices, and the lungs of immunocompromised cystic fibrosis patients (1). The infection processes are governed via complex posttranscriptional regulatory mechanisms stimulated by certain environmental stresses such as nutrient starvation and antibiotic exposure (2). Such posttranscriptional control networks are typically composed of globally acting RNA-binding proteins (RBPs) together with small non-coding RNAs (sRNAs). Considering the presence of a large number of putative sRNAs and widely conserved RBPs in P. aeruginosa, a number of them might be incorporated into the regulatory networks for the processes.
Host factor protein for the replication of phage qβ (Hfq) is one of the most well-known RBPs that acts as an RNA chaperone and helps sRNAs basepair with target mRNAs for repression or activation of the translations (3). Similar to Escherichia coli or Salmonella, Hfq contributes to varied phenotypes such as quorum sensing, virulence, and antibiotic resistance in P. aeruginosa (4–6). Likewise, the RNA chaperone CsrA family, originally discovered as a carbon storage regulator in E. coli, also plays a global role in posttranscription in many bacteria (7). Unlike Hfq, the CsrA family functions by repressing translation directly by competing with the ribosome rather than acting as a matchmaker between sRNA and mRNA in Gram-negative bacteria. CsrA family proteins bind to target RNAs via a stem-loop structure comprising an ANGGA motif. Conversely, translational suppression is inhibited by at least three sRNAs, namely, CrsB, CrsC, and McaS, each containing multiple GGA motifs in E. coli (8, 9).
P. aeruginosa has two CsrA homologs RsmA and RsmN (RsmA/N) and four redundant sRNAs RsmV, RsmW, RsmY, and RsmZ that function as antagonists of RsmA/N (10–12). RsmA and RsmN are structurally distinct with respect to the position of an α helix constituent; RsmA is composed of five consecutive β sheets and a C-terminal α helix whereas RsmN consists of an uniquely inserted α helix between the β2 and β3 sheets (13). RsmA/N-titrating sRNAs, RsmY/RsmZ (RsmY/Z), are regulated by GacA/GacS (GacAS) two-component system. When the transmembrane histidine protein kinase (HPK) GacS is activated by environmental signals involved in the transition to the stationary phase, it phosphorylates its cognate response regulator GacA (14). Upon phosphorylation, GacA promotes the transcription of RsmY/Z (15). While RsmW is activated in the stationary phase or by high temperature (11), the level of RsmV expression is relatively low during the whole course of the growth, independent of GacAS activity (12). Both RsmA/N bind to some, but not all, common regulatory targets via a conserved arginine residue. Most of the known RsmA/N targets such as genes encoding for type VI secretion systems (T6SS) and exopolysaccharide biosynthesis are subject to direct translational repression (16, 17). In contrast, the genes encoding motility and type III secretion systems (T3SS) are positively regulated by RsmA/N (14, 18), thus modulating the transition between acute and chronic infections in P. aeruginosa.
These relationships between RsmA/N and target genes have been extensively investigated by phenotypic assays combined with genetic analysis in vitro. However, the complexity of in vivo RNA-based regulations makes attaining a comprehensive understanding of the mode of actions of RsmA/N difficult. To address this, recent studies have identified target transcripts, that are bound by members of the CsrA/Rsm family via co-immunoprecipitation approaches (19, 20). Such methods can identify major RNA ligands. However, they do not provide sufficient information with respect to the RBPs binding sites within each transcript. Herein, we performed UV crosslinking immunoprecipitation followed by RNA-seq (UV CLIP-seq) with RsmA/N to identify their regulatory targets at a single-nucleotide resolution in vivo. Our approach found more than 500 potential binding sites genome-wide, which enabled us to gain new insights into the biological functions and the similarities and differences between the two redundant Rsm RBPs in P. aeruginosa.
RESULTS
Transcriptome-wide mapping of RsmA/N binding sites
As a preliminary investigation, western blotting analysis was performed using P. aeruginosa PAO1 chromosomal FLAG tagged strains: rsmA::3×FLAG, rsmN::3×FLAG, and rsmN::3×FLAGΔrsmA. Here, we constructed a third strain as RsmA negatively regulates the translation of rsmN (21). When compared with RsmA::3×FLAG protein, RsmN::3×FLAG was not detected despite the deletion of translational repressor rsmA (Fig. S1A). Therefore, we expressed the rsmN::3×FLAG cassette from a multicopy plasmid in the PAO1 wild type strain. RsmN was successfully detected by western blot when the medium was supplemented with the arabinose inducer at 0.1% concentration (Fig. S1B). In the following procedure, chromosomally tagged rsmA::3×FLAG strain and plasmid-inducible rsmN::3×FLAG strain were incubated during the early stationary phase (OD600 = 2.0) and UV irradiated to induce covalent bonds between RNAs and the corresponding proteins in vivo. Autoradiography and western blot analyses indicated that UV crosslinking and co-immunoprecipitation with anti-FLAG antibodies along with stringent washing enriched RsmA-RNA and RsmN-RNA complexes (Fig. 1A). It should be noted that we omitted RNA radiolabeling steps and cut membranes from the regions consisting of RsmA/N bands up to the region 50 kDa above for RNA purification (Fig. 1B).
Purified RNAs from crosslinked and non-crosslinked samples in triplicates were reverse transcribed into cDNA and subjected to Illumina sequencing. By performing a comparative analysis of called peaks from RsmA/N-binding regions between crosslinked and non-crosslinked libraries using DESeq2, we detected hundreds of binding sites with significant enrichment (false discovery rate (FDR) < 0.05 and minimum expression = 1.0) throughout the P. aeruginosa transcriptome (Fig. 1C). We identified 75 overlapping peaks between RsmA and RsmN, with overlapping peaks defined as the peaks that exhibited both the start and stop positions within 40 nt of each other, thus constituting 69 overlapping genes (Fig. 1D). Since this CLIP-seq was carried out in conditions similar to the previously performed Hfq CLIP-seq (22), the genes that overlapped between RsmA/N and Hfq were also investigated. A total of 25 genes including the genes encoding outer membrane proteins were common among all three RNA chaperones (Fig. S2A). Gebhardt et al. recently performed chromatin immunoprecipitation with cells grown in the presence or absence of rifampicin followed by high-throughput DNA sequencing (ChIPPAR-seq) for P. aeruginosa RsmA to capture a subset of nascent transcripts (20). When compared with the list of transcripts that are regulated co-transcriptionally by RsmA, approximately 25% of RsmA-binding genes were also detected in our CLIP-seq (Fig. S2B). In addition, Romero et al. mapped more than 500 transcripts directly bound by RsmN using RNA immunoprecipitation and sequencing (RIP-seq) (19). We observed that a limited number of RsmN-binding genes were detected in both our CLIP-seq and the RIP-seq (Fig. S2C).
Finally, significant peaks were classified on the basis of RNA classes using previously generated UTR annotation list (22–24) and manually curated P. aeruginosa sRNA lists (25–27). Most of the peaks were mapped within mRNAs, in which more than 40% of the binding sites in CDSes overlapped with the first quarter of the CDSes (Fig. 1E and Fig. S3A). Interestingly, approximately half of ncRNAs bound by RsmA were annotated as cis-antisense RNAs (asRNAs) (Fig. S3B), suggesting that certain posttranscriptional regulatory mechanisms attributed to RsmA and asRNAs are still unrecognized. Taken together, RsmA/N CLIP-seq analysis identified hundreds of binding sites associated with each RNA chaperone.
Similarities and differences in sequence and structural motifs bound by RsmA/N
RsmA/N bind to conserved GGA sequences within or close to Shine-Dalgarno sequences of target mRNAs (18). More precisely, parallel systematic evolution of ligands by exponential enrichment (SELEX) studies for RsmA/N have identified a common consensus motif CANGGAYG positioned in a hexaloop region of the stem-loop structure (28). To understand the consensus sequence bound by RsmA/N and its position in the target transcripts in vivo, the peak density of RsmA/N peaks across all detected mRNAs was determined via meta-gene analysis using start or stop codons as reference points. Strong peak densities were observed around start codons but not around stop codons, showing that P. aeruginosa RsmA/N preferentially bind to the 5’ UTRs of mRNAs (Fig. 2A). Next, sequence motifs for RsmA/N bindings were determined using all detected RsmA/N binding sequences by MEME motif analysis. Top-ranked motifs of both RsmA/N contained the ANGGA sequence (Fig. 2B). All RsmA binding sites exhibited the motif. In fact, more than 80% of detected RsmA/N peaks demonstrated at least one minimal GGA motif and the second nucleotide of ANGGA sequence for RsmA binding showed a preference for uracil (Fig. 2C). Unlike RsmA, the second nucleotide position of the ANGGA sequence for RsmN binding is more tolerant, accepting any nucleotides except for guanine (Fig. 2B and C). In order to understand whether the detected motifs were likely to be present in the apical loop, all detected RsmA/N binding sequences were subjected to CMfinder structural motif analysis. The top-ranked structural motifs from CMfinder were predicted as highly conserved stem-loop with the GGA sequence in the loop regions (Fig. 2D). Interestingly, the top-ranked structural motif for RsmN binding demonstrated two stem-loops with GGA sequences adjacent to each other, consistent with the previous report showing that RsmN requires two binding sites of the known target tssA1 (28). Therefore, the number of GGA sequences per peak was searched to evaluate whether RsmN requires two adjacent GGA sequences for high-affinity binding. Although we expected that RsmN might tend to bind peaks corresponding to more than two GGA motifs, no significant difference was observed between RsmA and RsmN (Fig. 2E). Altogether, RsmA/N CLIP-seq demonstrates ANGGA in loop regions as a general recognition sequence/structural motif, which is in clear accordance with previously published works.
RsmA/N CLIP-seq expands global binding targets in P. aeruginosa
When the small non-coding RNAs RsmY/Z are highly expressed, RsmA/N are titrated away from target RNAs playing an important role in the regulation of virulence factors associated with acute and chronic infections. The results of RsmA/N CLIP-seq conducted in this study demonstrate that the read coverage of RsmY/Z was highest among all other genes (Fig. 3A and Table S1). RsmY/Z exhibit multiple GGA motif sites for RsmA/N binding and RsmA/N peak sites corresponded to their GGA motifs (Fig. 3B), whereas Hfq predominantly associated with their Rho-independent terminators (29). Our data also shows that RsmA/N indeed bind to well-known target mRNAs. For examples, high CLIP-seq coverages were found at the 5’UTR of the gene tssA1 encoding structural component of the Hcp secretion island-I-encoded T6SS and 5’UTR of the gene mucA encoding anti-sigma factor (Fig. 3C). The RsmA/N-binding sites detected on these mRNAs fold into hairpins with GGA motifs (Fig. 3D). Collectively, the data suggests that RsmA/N CLIP-seq was able to capture the bona fide RsmA/N binding sites.
To determine the biological processes in which RsmA/N-binding RNAs were enriched, DAVID enrichment analysis was performed for mRNAs containing peaks, with a modified Fisher’s exact p-value threshold < 0.1. Genes associated with cell wall organization, including those involved in the polysaccharide biosynthetic process, O-antigen biosynthetic process, and lipopolysaccharide (LPS) biosynthetic process were enriched (Fig. 4A). Among the cell wall organization processes, high peak density was detected within the wbp gene cluster involved in O5 O-antigen biosynthesis (Fig. 4B). Since the category represents an unknown role for RsmA/N, we decided to explore them in more detail.
The wbp gene cluster consists of three genes responsible for the assembly of the O-antigen (wzx, wzy, and wzz), twelve genes involved in the biosynthesis assembly of the nucleotide sugars of the O unit (wbpABCDEGHIJKLM), three insertion genes encoding putative transposase and integrase (PA3142–PA3144) between wbpL and wbpM, and two non-LPS-related genes (hisF2 and hisH2) between wbpG and wzx (30). Significant peaks indicating RsmA binding were observed in wzz, wbpA, wbpB, hisH2, hisF2, wbpG, wbpH, wbpL, and wbpM genes whereas those indicating RsmN binding were observed in wbpA, wbpE, wbpI, and wbpM genes (Fig. 4C). We verified the function with respect to RsmA/N-mediated gene regulation against wzz, wbpA, wbpE, wbpG, wbpH and wbpM using super-folder GFP (sfGFP) translational fusion assay. When compared with the wild type, ΔrsmA/N strain expressed significantly high GFP fluorescence in wzz, wbpA, wbpE, and wbpG translational fusions, suggesting that RsmA/N posttranscriptionally repress the translation of each gene (Fig. 4D). Altogether, our RsmA/N CLIP-seq results reveal new RsmA/N regulatory targets associated with LPS modification.
Homeostasis of RsmY/Z expressions by an RsmA/N-controlled feedback loop
The expression of RsmA/N-titrating sRNAs RsmY/Z is activated by GacAS two-component system (15). The GacAS system is controlled by three additional HPKs: RetS, PA1611, and LadS. In addition to these complex regulatory pathways, RsmY and RsmZ are independently regulated by the histidine phosphotransfer protein HptB and cytoplasmic axial filament protein CafA, respectively. Expression of hptB is regulated by additional membrane-binding proteins SagS and ErcS (31), whereas the cafA expression is under the control of another two-component system BfiR/BfiS (32). Among those proteins involved in the homeostasis of RsmY/Z expression, we observed RsmA/N binding peaks in the sagS, gacS, ladS, hptB, and cafA genes (Fig. 5A). Since HptB is the decision hub for RsmY activation via the phosphorylation of additional regulatory components HsbA/HsbD and flagellar gene expression via FlgM sequestration (33), we focused on RsmA/N-mediated hptB gene regulation. We also investigated whether RsmA/N directly regulated cafA gene expression since CafA specifically represses RsmZ expression with its endoribonucleolytic activity.
We observed RsmA/N binding peaks in both coding DNA sequences although only RsmN binding was statistically significant (Fig. 5B). Therefore, we performed the sfGFP translational fusion assay to understand the RsmA/N associated gene regulation of hptB and cafA. When compared with the wild type, ΔrsmA/N strain expressed significantly high GFP fluorescence in both translational fusions (Fig. 5C). In order to investigate whether both RsmA and RsmN repress the hptB and cafA translations, RsmA and RsmN were exogenously expressed from multicopy plasmid pJN105 in ΔrsmA/N background and GFP fluorescence was measured. When compared with pJN105 vector control, both RsmA/N were capable of repressing GFP fluorescence (Fig. 5D). If hptB expression is negatively regulated by RsmA/N, downstream HptB-dependent secretion and biofilm anti anti-sigma factor HsbA should be phosphorylated and diguanylate cyclase HsbD should activate RsmY expression. Additionally, if cafA expression is negatively regulated by RsmA/N, degradation of RsmZ by CafA should be alleviated. To confirm these, RsmY/Z abundance between the wild type and ΔrsmAN strains was quantified by qRT-PCR. As expected, RsmY/Z were significantly reduced in the ΔrsmAN strain (Fig. 5E), suggesting that RsmY/Z expressions were balanced by RsmA/N-controlled feedback loop.
RsmA/N bind to multiple sRNAs other than RsmY/Z in P. aeruginosa
Besides RsmY/Z, we observed high read coverage for RsmA/N bindings and significant enrichment of crosslinked samples in several sRNAs. For example, SPA0035 sRNA, a transcript from the minus strand of the intergenic region between ada and PA2119, was significantly bound to RsmA/N, but not to Hfq (Fig. 6A, left). As another example, the PAO1-specific asRNA SPA0066 transcribed from the opposite strand of PA3993 was also significantly bound to RsmA/N, but not to Hfq (Fig. 6A, middle). It should be noted that SPA0066 overlapped in the antisense orientation of the 5’ UTR of a gene encoding a putative transposase of the IS116/IS110/IS902 family. This transposase family exists in six genomic positions in PAO1, all of which demonstrate identical asRNAs (SPA0004–8 and SPA0066). The SPA0079 sRNA, a transcript from the minus strand of the intergenic region comprising 1,011 nt between PA2763 and PA2764, was significantly bound to RsmA/N, as well as to Hfq (Fig. 6A, right and Fig. S4A). The majority of these sRNAs carry GGA motifs and many corresponding peaks fold into hairpins (Fig. 6B).
Among the RsmA/N-binding sRNAs detected in our CLIP-seq, SPA0079 exhibited significant binding to both Hfq and RsmA/N and demonstrated high coverage next to RsmY/Z (see y-axis in Fig. 6A and Table S1). Subsequently, we investigated its characteristics as an RsmA/N-binding sRNA in next section.
SPA0079 sRNA does not sequester RsmA/N away from target mRNAs under our conditions
We first determined the SPA0079 boundaries by 5’/3’ RACE analysis. RNA pyrophosphohydrolase RppH treatment followed by PCR amplification selectively enriched 5’ terminus with triphosphate ends of SPA0079 (the band indicated by a red arrow in Fig. S4B). The 3’ terminus of SPA0079 corresponded to the canonical U repeats of the rho-independent terminator (Fig. S4A). The SPA0079 secondary structure predicted by Mfold (34) exhibited only a single stem-loop, having a strong resemblance to a rho-independent transcription terminator (Fig. S4C). The expression of SPA0079 in the wild type and the deletion mutant were measured throughout the course of growth (Fig. S4D). The maximal level of the SPA0079 expression was reached in the late stationary phase at the OD600 of 6.0 in the wild type (Fig. S4D, lanes 1–5). In contrast, SPA0079 sRNA level was undetectable in the ΔSPA0079 mutant strain (Fig. S4D, lane 6).
A previous study has suggested that two quorum sensing (QS) pathways LasI/LasR and RhlI/RhlR might be related to the expression of SPA0079 (35). Furthermore, the DNA sequence of the upstream of −35 promoter motif was similar to the consensus motif of LasR/RhlR binding (Fig. S4A). To investigate QS-dependent SPA0079 expression, northern blotting analysis was performed with ΔlaslΔrhlI double mutant strain with or without the exogenous supplementation of two acyl homoserine lactones (AHLs) 3OC12-HSL and C4-HSL. SPA0079 displayed strict dependence on QS as it accumulated within 1 h after AHLs were added (Fig. S5A). Additionally, SPA0079 was not detectable in a ΔlasI strain, whereas it was detected moderately in a ΔrhlI strain (Fig. S5B). Taken together with the published report (35), these results suggest that the expression of SPA0079 is controlled by the QS systems. Another previous report showed that Hfq stabilizes RsmY by blocking the cleavage by RNase E (29). We investigated whether Hfq might affect SPA0079. Northern blotting showed no difference in the SPA0079 levels in a Δhfq mutant compared to its levels in wild type and ΔrsmA/N mutant (Fig. S5C).
To investigate whether SPA0079 indeed titrates RsmA/N away from the mRNA target, the activity of sfGFP fused with 5’ UTR and CDS of cafA was assayed with or without SPA0079 overexpression. Although we speculated that a cafA::sfGFP fusion would be repressed by the induction of SPA0079, no clear difference was observed in GFP activity in the SPA0079 overexpression state compared to that in the ΔrsmA/N mutant strain (Fig. S5D). Considering that the level of SPA0079 expression reaches maximum in the late stationary phase (Fig. S4D), and that RsmY/Z predominantly bind to RsmA/N under the conditions in which CLIP-seq was performed, it may be that SPA0079 sRNA competes or complements RsmY/Z under specific conditions not captured in our assay. Future studies are required to determine whether or not SPA0079 is a functional RsmA/N antagonist.
DISCUSSION
Two conserved and redundant RBPs RsmA/N play a critical role in balancing acute and chronic infections in P. aeruginosa. So far, the interactions of RsmA/N with target RNAs have been extensively explored in vitro. However, it is prudent to understand the mode of actions in the complex transcriptional networks in vivo. Many studies using advanced technologies based on high-throughput sequencing have now provided the global regulatory functions of RBPs depending on the physiological states of a cell (36–38). In this study, we performed the RsmA/N CLIP-seq analysis and demonstrated more than 500 genome-wide RsmA/N binding sites. Many genes identified in this study could be attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.
As the prerequisite for this study, we first checked RsmA and RsmN abundance at an OD600 = 2.0. It is well known that the transcript encoding RsmN is posttranscriptionally repressed by RsmA and the protein abundance is relatively low compared to RsmA during the growth period (21). Therefore, we speculated that RsmN-binding RNAs are likely captured in the ΔrsmA background. However, the abundance of RsmN is still low even in the ΔrsmA background and interacting RNAs cannot be captured by co-immunoprecipitation (Fig. S1). The low RsmN abundance may be attributed to its original low transcription rate in the condition where our CLIP-seq was conducted. A previous study has shown that the level of rsmN expression reaches maximum in the late stationary phase (39). Although we relied on the exogenous expression of RsmN from multicopy plasmid and performed RsmA/N CLIP-seq at an OD600 = 2.0 in order to compare previously performed Hfq CLIP-seq result (22), it would be possible to detect RsmN-binding positions using the chromosomally tagged strain at the later growth phase, which should be addressed in further experiments.
In the widespread application of the current CLIP-seq procedure, radioactive labeling of crosslinked RNAs and visualization of the autoradiograph are common. As with several non-radioactive CLIP methods (40–43), herein, we omitted labor-intensive use of radioactive substances and successfully captured specifically interacting RNAs (Fig. 1B to E). When compared with previously published data, RsmA/N-binding genes demonstrated in this study were relatively low in number (Fig. S2). This variation may be attributed to the differences in the experimental setup. RsmA ChIPPAR-seq captures nascent transcript at mid-log phase and significant binding positions are defined as enrichment in the minus rifampicin library compared with the plus rifampicin library (20). RsmN RIP-seq is performed during the late stationary phase and significant binding genes are defined using enrichment index generated by comparison of co-immunoprecipitation library with total RNA library (19). In addition, the small overlap of detected genes with previous reports perhaps derives from the limitations of bioinformatics analysis. Our CLIP-seq approach relies on the comparison between crosslinking and non-crosslinking libraries. Hence, this approach identifies the binding sites with significant enrichment in crosslinking samples. The analysis perhaps missed the bona fide RsmA/N binding sites with high-affinity in non-crosslinking libraries. Especially in our RsmA CLIP-seq, the background likely has much higher counts than that specific to the crosslinking libraries, which might be the artifact of one or two highly expressed transcripts such as RsmY and RsmZ (Fig. 3A and Fig. S6). Nevertheless, numerous known targets of RsmA/N were observed from our CLIP-seq (Fig. 1). As examples, we showed high CLIP-seq coverages observed at the 5’UTRs of the mRNAs tssA1 and mucA and sRNAs RsmY/Z, leading to the identification of their RsmA/N-binding sites in vivo at a single-nucleotide resolution (Fig. 3).
Interestingly, approximately half of RsmA-binding ncRNAs were annotated as asRNAs (Fig. S3B). Although no report exists demonstrating the promotion of the complex formation between regulatory RNAs and their target mRNAs in Gram-negative bacteria by the CsrA family, Bacillus subtilis CsrA helps SR1 sRNA basepair with ahrC mRNA, affecting the expression of its downstream genes (44). Rather than the CsrA family, ProQ/FinO-domain proteins and Hfq are generally thought to be responsible for the interaction between mRNAs and asRNAs in Gram-negative bacteria (45). The E. coli F plasmid-encoded FinO promotes the association of F plasmid-encoded mRNA traJ with asRNAs FinP (46). The chromosomally encoded ProQ from Salmonella also promotes the association of mRNA with asRNAs including Type I Toxin-Antitoxin system (47, 48), although ProQ can also play a similar role in Hfq acting as a matchmaker between trans-encoded sRNAs and mRNAs (49). Hfq primarily promotes the baseparing between trans-encoded sRNAs and mRNAs. However, it also facilitates antisense paring such as in IS10 system, where asRNA RNA-OUT interacts with RNA-IN encoding transposase to occlude the ribosome binding site (50). Since we found that asRNAs SPA0004–0008 and SPA0066 transcribed from the opposite strands of genes encoding putative transposases associate with RsmA/N (Fig. 6A, middle), it should be interesting to investigate whether these asRNAs may regulate the opposite genes via RsmA/N binding similar to Hfq, RNA-IN, and RNA-OUT in E. coli.
The advantage of CLIP-seq is to detect protein binding sites on the transcript at a single-nucleotide resolution. This approach has demonstrated sequence/structural binding motifs of the CsrA family in E. coli and Salmonella (37, 38). Herein, we investigated the similarities and differences between P. aeruginosa RsmA and RsmN on the basis of sequence/structural motifs and a typical distribution of the detected peaks (Fig. 2). The results of the peak distribution among mRNAs were common between the two RBPs, indicating that RsmA/N peaks are highly enriched at only 5’ UTRs, which is consistent with the previous reports (19, 51) (Fig. 2A to D). In addition, sequence and structural motif analyses demonstrated that the stem-loop structure with the ANGGA sequence in its loop region is primary of RsmA/N bindings. Interestingly, although all of the RsmA peaks detected in this study include the ANGGA motif, RsmN peaks seem to be under looser definition of its binding motif than RsmA (see the number of detected peaks in Fig. 2B). From the perspective of molecular basis, RsmA homolog RsmE in Pseudomonas fluorescens CHA0 needs the looped out nucleotide base N in ANGGA motif and baseparing in the stem for rigid binding through Leu55 to Ala57 in the C-terminal α helix as well as the correct stacking (52). In contrast, α helix in RsmN is located within the internalized β2 and β3 sheets, constituting the hydrophobic core that acts as a potential new RNA binding site (13). Thus, RsmN may bind with a more flexible loop motif independent of C-terminal α helix even without obvious ANGGA sequence. Interestingly, the structural motif of RsmN peaks consisted of two tandem stem-loop structures with GGA motifs (Fig. 2D). Consistent with this, in vitro SELEX study and mutational analysis have demonstrated long RsmA/N targets with two consensus GGA motifs, and both of the two tandem GGA sites are required only for RsmN binding (28). Overall, our CLIP-seq analysis combined with previous reports suggested a unique two-sidedness of RsmN binding; tandem stem-loop structures with two GGAs or flexible sequences without obvious ANGGA sequence.
Further genetic analysis has elucidated new regulatory targets of RsmA/N involved in O5 O-antigen biosynthesis (Fig. 4). The three genes wzx, wzy, and wzz are responsible for the assembly of the O-antigen and other genes wbpABCDEGHIJKLM are involved in the biosynthesis assembly of the nucleotide sugars of the O unit (30). Since we observed RsmA bindings to both wzz and wzz2 encoding O-antigen chain length regulators and validated the RsmA/N-mediated translational repression of wzz (Fig. 4D and Table S1), active posttranscriptional regulation through RsmA/N would lead to LPS-rough phenotype, which is often observed in P. aeruginosa isolates from chronic pulmonary infections (53, 54). In a recent study, the low levels of wzz2 expression led to the conversion to mucoid phenotype with alginate production, and a transcriptional factor AmrZ negatively regulated the expression (55). Under the same AlgU regulon with AmrZ, AlgR activates alginate production and in turn, directly stimulates RsmA expression (56). This might accelerate the modulation of O-antigen length and eventually the biofilm formation (57). The translational repression of genes involved in O-antigen biosynthesis through RsmA/N is also consistent with the findings demonstrating that the absence of B-band O-antigen is correlated with the increase of T3SS-mediated cytotoxicity that is activated by RsmA/N directly (58). Taking into account the previous ChIP-seq analysis showing that T3SS master regulator ExsA binds to promoter sequences of wbpA and wbpH (59), the wbp gene cluster is likely to be under the both transcriptional and translational regulation mediated by RsmA/N.
Free RsmA/N and the level of the titrating sRNAs are controlled through homeostatic regulation. Although it was previously shown that RsmA exerts a positive effect on RsmY and RsmZ transcription in P. aeruginosa and Pseudomonas protegens (60, 61), the mechanisms underlying the positive feedback loop remain unclear. In this study, hptB encoding the histidine phosphotransfer protein and cafA encoding the cytoplasmic axial filament protein were identified as novel RsmA/N regulatory targets (Fig. 5). When RsmA/N repress the translation of hptB, RsmY expression would be stimulated via the phosphorylation of HsbA and the subsequent activation of HsbD (31). In addition to RsmY, when the cafA is repressed in the RsmA/N-dependent manner, RsmZ would escape from degradation owing to the endoribonucleolytic activity (32), thus constituting three repressors feedback loop. In a simple and synthetic gene circuit, however, the three repressors loop periodically induces the synthesis of output (62). Since RsmY/Z expression increases throughout the growth period (15), other factors might be involved in the homeostatic regulations of RsmY/Z. For example, polynucleotide phosphorylase PNPase regulates the stabilities of RsmY/Z (63). Our CLIP-seq data has identified RsmA binding to the gene encoding PNPase (Table S1), which infers that RsmA affects RsmY/Z through the availability of PNPase.
Finally, we discovered 41 RsmA/N-binding sRNAs besides known RsmA/N-titrating sRNAs (Fig. 6 and Table S1). Among them, SPA0079 was highly enriched in both Hfq and RsmA/N CLIP-seq, motivating us to further investigate its properties. The level of SPA0079 expression reached maximum in late stationary phase and was undetectable in ΔlasIΔrhlI mutant, whereas exogenous supplementation of two AHLs complimented the expression (Fig. S4 and S5), strongly supporting the fact that SPA0079 is regulated by two QS pathways (35). Although we expected that SPA0079 could alleviate RsmA/N-mediated translational repression, no clear activation of translational fusion activity was observed with the SPA0079 sRNA overexpression (Fig. S5D). Given that RsmY/Z predominantly bind to RsmA/N under the conditions in which CLIP-seq was performed and the effect of SPA0079 is negligible, future studies should be performed using ΔrsmY/Z mutant to determine whether or not the SPA0079 sRNA are indeed RsmA/N antagonist. A recent paper shows that sRNA179 (also annotated as SPA0034) expression stimulates RsmY transcription (64). Additionally, the expression of sRNA179 is activated by QS similar to SPA0079 (35). Considering these observations, QS might suppress the RsmA/N regulatory system through two different posttranscriptional pathways, highlighting the complexity of Rsm regulatory systems.
METHODS
Strains, plasmids, and growth conditions
Strains, plasmids and oligonucleotides used herein are enlisted in Table S2. All experiments were performed using P. aeruginosa PAO1 or its derived strains. Each strain was cultured at 37°C in Luria-Bertani (LB) medium. Samples were collected at the OD600 values indicated in the figures. Where indicated, the appropriate AHLs were added at the following final concentrations: C4-HSL (Cayman Chemicals) at 10 μM and 3OC12-HSL (RTI International) at 2 μM. Antibiotics and arabinose were used as needed at concentrations listed as follows. For Pseudomonas, gentamicin, tetracycline, and arabinose at concentrations 50 μg/ml, 80 μg/ml, and 0.1%, respectively, were used. Similarly, for E. coli, gentamicin and tetracycline at concentrations 10 μg/ml and 20 μg/ml, respectively, were used.
Strain construction
P. aeruginosa PAO1 ΔrsmA, ΔrsmN, ΔrsmA/N double mutant, ΔlasI, ΔrhlI, ΔlasIΔrhlI double mutant, and ΔSPA0079 strains were constructed based on the conjugative transfer of appropriate plasmids and homologous recombination between chromosome and plasmids as described previously (65). The plasmids for the homologous recombination of ΔrsmA, ΔrsmN, and ΔrsmA/N double mutant were constructed by HindIII/XbaI cloning of PCR products 500 bp upstream and 500 bp downstream of CDS of each gene from PAO1 chromosome into pG19II backbone (66). The plasmids for the homologous recombination of ΔlasI, ΔrhlI, ΔlasIΔrhlI double mutant, and ΔSPA0079 were constructed by HindIII/BamHI cloning of PCR products 500 bp upstream and 500 bp downstream of CDS of each gene from PAO1 chromosome into pG19II backbone (66). pG19rsmA::3×FLAG and pG19rsmN::3×FLAG were constructed by cloning PCR products at HindIII/XbaI sites 500 bp upstream of the rsmA/N stop codon in the PAO1 chromosome, 3×FLAG tag, 500 bp downstream of the rsmA/N stop codon in the PAO1 chromosome into the pG19II backbone.
UV crosslinking, immunoprecipitation, and RNA purification
Bacterial cultures with volume 200 ml of three replicates were maintained up to an OD600 of 2.0 in LB medium. For RsmN expression from the multicopy plasmid, 0.1% arabinose was added at an OD600 of 0.8. UV crosslinking and immunoprecipitation were performed in accordance with the previously published protocol with minor modifications (22). Briefly, half of the culture at an OD600 of 2.0 from each condition was irradiated at 800 mJ of UV light at 254 nm. After UV crosslinking, the sample was centrifuged at 3,600 rpm for 30 min at 4°C along with the non-crosslinked control samples.
Cell pellets were lysed in FastPrep 24 (MP-Biomedicals) at 6 m/s for 1 min with 1 ml of 0.1-mm glass beads and 800 μl NP-T buffer (50 mM NaH2PO4, 300 mM NaCl, 0.05% Tween20, pH 8.0). NP-T supplemented with 8 M urea was added to each supernatant at an equal volume and incubated for 5 min at 65°C with shaking at 900 rpm. Anti-FLAG magnetic beads were washed thrice with 500 μl NP-T buffer, resuspended in 125 μl NP-T buffer, and treated with a 120-μl suspension of urea-treated samples. After 1 h of incubation at 4°C, samples were washed twice with 500 μl high-salt buffer (50 mM NaH2PO4, 1M NaCl, 0.05% Tween20, pH 8.0), followed by two washes with 500 μl NP-T buffer. Beads were resuspended in benzonase mix [500 units benzonase nuclease (E1014, Sigma-Aldrich) in NP-T buffer with 1 mM MgCl2] and incubated for 10 min at 37°C with shaking at 900 rpm. After one wash with high-salt buffer and two washes with CIP buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2), beads were resuspended in 200 μl CIP mix (20 units of calf intestinal alkaline phosphatase (M0290, NEB) in CIP buffer) and incubated for 30 min at 37°C with shaking at 800 rpm.
Here, radioactive isotope (RI) labeling was performed only in the preliminary investigation to check whether UV crosslinking did enrich RNA-protein complex (Fig. 1A). After one wash with high-salt buffer and two washes with PNK buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1 mM spermidine), beads were resuspended in 200 μl PNK buffer and separated into 20 μl and 180 μl volumes to be used subsequently for western blotting and RI labeling, respectively. For RI labeling, beads were magnetized and resuspended in 100 μl PNK mix [10 units of T4 polynucleotide kinase (EK0032, ThermoFisher Scientific), 10 μCi γ-32P-ATP in PNK buffer]) and incubated for 30 min at 37°C, following the addition of 1 μl 10 mM non-radioactive ATP and incubation for 5 min at 37°C. After two washes with NP-T buffer, beads were resuspended in 25 μl 2xProtein loading buffer and incubated for 5 min at 95°C. Beads were magnetized and supernatants were transferred to fresh tubes. The elution was repeated twice. For western blotting, 20 μl separated beads were washed twice with 100 μl NP-T buffer and resuspended in 10 μl 2xProtein loading buffer and incubated for 5 min at 95°C. Beads were magnetized and supernatants were transferred to fresh tubes. The elution was repeated twice.
In the CLIP experiments for RNA purification and cDNA preparation, RI labeling was not used (Fig. 1B). After CIP reaction, beads were resuspended in 100 μl PNK mix [10 units of T4 polynucleotide kinase (EK0032, ThermoFisher Scientific), 1μl 10 mM non-radioactive ATP in PNK buffer] and incubated for 30 min at 37°C, followed by addition of 1 μl 10 mM non-radioactive ATP and incubation for 5 min at 37°C. After two washes with NP-T buffer, beads were resuspended in 30 μl 2xProtein loading buffer and incubated for 5 min at 95°C. Beads were magnetized and supernatants were transferred to fresh tubes. The elution was repeated twice.
Aliquots of 55 μl were loaded and separated via SDS-polyacrylamide gel electrophoresis (12% resolving gel) at 30 mA while moving through the stacking gel, which was then increased to 40 mA. After RNA electrophoresis, the RNA was transferred from the gel to the Protran membrane (#10600016, GE Healthcare). The membrane was placed on a clean glass surface and cut from the prestained protein markers to 50 kDa above them. Each membrane piece was cut into smaller pieces and placed in 2 ml fresh tubes with 400 μl PK solution [1.3 mg/ml Proteinase K (EO0491, ThermoFisher Scientific), and 10 units of RNase inhibitor (#10777019, Invitrogen) in 2xPK buffer (100 mM Tris-HCl, pH 7.9, 10 mM EDTA, 1% SDS)], followed by incubation for 1 h at 37°C with shaking at 1,000 rpm. The samples were subsequently incubated with 100 μl PK buffer containing 9 M urea for 1 h at 37°C with shaking at 1,000 rpm. Thereafter, 450 μl of supernatants from proteinase K-treated membranes were mixed with an equal volume of phenol:chloroform:isoamyl alcohol in phase-lock gel tubes and incubated for 5 min at 30°C with shaking at 1,000 rpm. Each mixture was centrifuged for 12 min at 13,000 rpm at 4°C, and 400 μl of the aqueous phase was precipitated with 3 volumes of ice-cold ethanol, 1/30 volume of 3 M NaOAc (pH 5.2), and 1 μl of GlycoBlue (AM9515, Invitrogen) for 2 h at −20°C. The precipitated pellet was washed with 80% ethanol, briefly dried for 5 min at 20°C, and resuspended in 11 μl of sterilized water.
cDNA library preparation and sequencing
A cDNA library of the CLIP samples was prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (#E7300, NEB) in accordance with the manufacturer’s instructions. RT primer and both 3’/5’ SR adapters were diluted 10-fold with nuclease-free water before use. cDNA was converted from 2.5 μl of each purified RNA. Reverse-transcribed cDNAs were amplified by 20 cycles PCR. PCR products were concentrated to 10 μl using the MinElute PCR Purification kit and separated by 6% polyacrylamide gel with 7 M urea. Bands between 130 bp and 250 bp were cut and purified from the gel in accordance with the manufacturer’s instructions. Purified cDNAs of 5 μl volume were amplified by 6 cycles PCR and concentrated to 10 μl using the MinElute PCR Purification kit. Final cDNA libraries were quantified using the Qubit dsDNA assay (Q32854, Invitrogen) and Agilent 2100 Bioanalyzer DNA HS (Agilent). Twelve cDNA libraries from CLIP were pooled on an Illumina HiSeq2500 and sequenced in paired-end mode (2 × 50 cycles).
Sequence processing, mapping, normalization, and peak calling
To assure high sequence quality, read 1 (R1) and read 2 (R2) files containing the Illumina paired-end reads in FASTQ format were quality and adapter-trimmed by Cutadapt (67) version 1.15/1.16 using a cut-off Phred score of 20. Reads without any remaining bases were discarded (command line parameters: −q 20 −m 1 −a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-A GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT). To eliminate putative PCR duplicates, paired-end reads were collapsed using FastUniq (68) After trimming, we applied the pipeline READemption (69) version 0.4.5 to align all reads longer than 11 nt to the P. aeruginosa PAO1 chromosome (NCBI accession no. NC_002516.2) reference genome using segemehl (70) version 0.2.0 with accuracy cut-off of 80%.
Read counts per position were analyzed using reads that mapped uniquely to single genomic position. The core of positions present in crosslinked and non-crosslinked library pairs were isolated, as described previously (71). Positions with low read counts were filtered from both crosslinked and non-crosslinked libraries using 10 standard deviations from 0 as a threshold. After plotting the difference in read counts between the two libraries as shown in Fig. S6, the size factor was calculated using the DESeq normalization procedure from the high-count positions in both libraries across all replicates (72).
We applied PEAKachu v0.1.0 (https://github.com/tbischler/PEAKachu) for the peak calling similar to the previously described protocol (71). First, BAM files for the respective pairs of crosslinked and non-crosslinked libraries were used to run in paired-end (-P) and paired-replicates (-r) mode. The maximum fragment size (-M) was set to 50 and annotations generated as GFF format were used to map overlapping features to the called peaks. Normalization was performed in the ‘‘manual’’ mode using previously determined size factors (see above). Other parameters were set to default. Second, the boundary of initial peaks was set through block definition computed by the blockbuster algorithm (73) based on pooled read alignments from all crosslinked libraries using default parameters. Third, the PEAKachu tool ran the DESeq2 package (74) to analyze the significance of peak enrichment in the crosslinked libraries relative to the non-crosslinked libraries with parameter values as follows: mad-multiplier (-m) 1.0, fold change (-f) 1.0, and adjusted p-value (-Q) 0.05. Finally, PEAKachu was used for each replicon and strand to generate normalized coverage plots to facilitate data visualization.
Analysis of sequence and structural motifs
The sequences of peaks were used to perform MEME sequence motif analysis (75). Minimum and maximum motif widths were set at 6 and 50, respectively, while other parameters were set to default. Structural motifs of the sequences of peak regions extended by an additional 10 nt upstream and downstream were analyzed using CMfinder 0.2.1 (76). The minimum length of single stem-loop candidates was set to 20, while other parameters were set to default. Each analyzed motif was visualized using R2R (77).
Statistical and other analysis
Descriptive statistical analyses for peak and gene overlapping between RsmA and RsmN, peak distributions across the P. aeruginosa genome, and peak classification among RNA classes were performed using Microsoft Excel. Meta-gene analyses across detected mRNAs or the P. aeruginosa genome were performed with in-house script using Python3. Genes identified via CLIP-seq analysis were functionally characterized using the PseudoCAP annotation (http://www.pseudomonas.com). KEGG enrichment analysis was performed for mRNAs identified via CLIP-seq analysis using DAVID (https://david.ncifcrf.gov/summary.jsp). Default parameters and databases were used for the analysis.
Construction of translational fusion plasmids
The sequence containing the first 186 amino acids of LacZ (LacZ186) and the super-folder GFP (sfGFP) open reading frame (78) expressed by J23119 constitutive promoter was obtained from ThermoFisher Scientific. LacZ186 was flanked by a NsilI and a SphI site while sfGFP was flanked by a SphI and an XbaI site. The synthetic sequence was cloned into inversely amplified pSW002-Pc PCR product by infusion cloning (Takara, Z9633N). pSW002-Pc was a gift from Rosemarie Wilton (Addgene plasmid # 108234) (79). The region from TSSes to downstream of the start codon in target RNAs was amplified with 15 bp complemented sequences on 5’ and 3’ end, respectively. The resulting PCR products were cloned into a NsilI/SphI digested pSW-lacZ::sfGFP backbone by infusion cloning (Takara, Z9633N).
Translational fusion assay
P. aeruginosa wild type and ΔrsmA/N strains carrying the sfGFP translational fusions were grown overnight in 1 ml LB medium with 80 μg/ml tetracycline at 37°C. An aliquot with 2 μl volume was resuspended in 200 μl LB medium with 80 μg/ml tetracycline and transferred to black polystyrene 96-well microplates with a clear, flat bottom (Corning). The medium was additionally supplemented with gentamicin with concentration 50 μg/ml and 0.1% arabinose for RsmA/N and SPA0079 expression from multicopy plasmids. Fluorescence polarization (FP476/510) was measured with SpectraMax GeminiXS (Molecular Devices) every 15 min for 6 h at 37°C with agitation for 1 min before fluorescence measurement. The fluorescent cutoff was set to 495 nm. Thereafter, the final optical density (OD) of each culture was obtained by GeneQuant 1300 (Biochrom). GFP activity was expressed in arbitrary units as FP476/510/OD600.
qRT-PCR
Total RNA was extracted by the hot phenol extraction method followed by DNase treatment. cDNAs from 1 μg DNase-treated purified RNA were obtained using PrimeScriptTM RT Master Mix (#RR036A, TAKARA) following the manufacturer’s instruction. qRT-PCR was performed for triplicates. Reactions were performed in a total volume of 20 μl containing 10 μl of TB Green Fast qPCR Mix (#RR430A, TAKARA), 0.4 μl of ROX reference dye II, 0.8 μl of each primer (10 μM), 7 μl of RNase-free water and 1 μl of template DNA. PCR and fluorescence measurements were performed using the viiA7 (Applied Biosystems) with the following program; preheating at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 20 s. The gene rpoD was used as an internal standard (80). Relative gene expression was calculated using the ΔΔCt method (81).
5’ and 3’ rapid amplification of cDNA ends (RACE)
5’ and 3’ RACE assays were performed as previously described with some modifications (82). For 5’ RACE assay, 15 μg of DNA-depleted RNA was incubated with 12.5 units of RNA 5’ pyrophosphohydrolase (RppH) (#M0356, NEB) in a 50 μl reaction for 1 h at 37°C. The same volume of RNA without the RppH reaction was prepared to be used as a negative control. After incubation, RppH-reacted and control RNAs were purified and 1.25 μM 5’RACE adaptor (5’-GAU AUG CGC GAA UUC CUG UAG AAC GAA CAC UAG AAG AAA-3’) was mixed. The samples were denatured at 95°C for 5 min. The adaptor was ligated using T4 RNA Ligase 1 overnight at 16°C. The adaptor-ligated RNA was purified, annealed with gene-specific primer and reverse-transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (#600107, Agilent) with following conditions; 42°C for 20 min, 55°C for 20 min and 70°C for 15 min in a 20 μl reaction. One-micro-liter of reverse-transcribed cDNA was amplified by nested PCR with a total volume of 50 μl containing 0.4 μM of 5’adaptor-specific primer, 0.4 μM of 1st round gene-specific primer, 200 μM of each dNTP, 1x PCR buffer, and 1.25 units of TaKaRa Taq HS (#R007A, TAKARA). Second PCR was performed with a total volume of 50 μl containing 1 μl of PCR product obtained after the first round, 0.4 μM of 5’adaptor-specific primer, 0.4 μM of 2nd round gene-specific primer, 200 μM of each dNTP, 1x PCR buffer, and 1.25 units of TaKaRa Taq HS (#R007A, TAKARA). Following conditions were used for both PCRs: preheating at 95°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and then final extension at 72°C for 7 min.
For 3’ RACE assay, 15 μg of DNA-depleted RNA was incubated with 25 units of calf intestinal alkaline phosphatase (CIP) (#M0290, NEB) in a 50 μl reaction volume for 1 h at 37°C. After incubation, RNA reacted with CIP was purified and 1.25 μM 3’RACE adaptor (5’-phosphate-UUC ACU GUU CUU AGC GGC CGC AUG CUC-idT −3’) was mixed. The samples were denatured at 95°C for 5 min. The adaptor was ligated overnight using T4 RNA Ligase 1 at 17°C. The adaptor-ligated RNA was purified, annealed with gene-specific primer and reverse-transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (#600107, Agilent) with following conditions; 42°C for 20 min, 55°C for 20 min and 70°C for 15 min in a 20 μl reaction. Reverse-transcribed cDNA of 1 μl was amplified by PCR in a total volume of 50 μl containing 0.4 μM of 3’adaptor-specific primer, 0.4 μM of gene-specific primer, 200 μM of each dNTP, 1x PCR buffer, and 1.25 units of TaKaRa Taq HS (#R007A, TAKARA). The PCR was conducted as follows: preheating at 95°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and then final extension at 72°C for 7 min.
PCR products were separated on 4% NuSieve GTG agarose gel, the band of interest was cut, gel-eluted, and cloned into a BamHI/HindIII digested pUC19. Bacterial colonies obtained after the transformation were screened by colony PCR. The PCR fragments were purified by QIAquick PCR purification kit and sequenced with an ABI Genetic analyzer 3500 (Applied Biosystems).
Northern blotting
Purified RNA of 10 μg was denatured at 95°C for 5 min in gel loading buffer II (#AM8547, Invitrogen) and separated by 6% polyacrylamide gel with 7M urea at 300 V for 2 h. Separated RNA was electro-transferred from the gel to Hybond-N+ membranes (GE Healthcare) at 50 V for 1 h at 4°C and the membranes were UV-crosslinked (120 mJ/cm2). Northern blotting was performed with the Roche DIG system. DNA probes for 5S rRNA and SPA0079 were amplified with PCR using primers described in Table S2 and PCR DIG Probe Synthesis Kit (#11636090910, Roche). The reaction was performed with a total volume of 50 μl containing 0.2 μM of forward primer, 0.2 μM of reverse primer, 200 μM of dATP, dCTP, and dGTP, 130 μM of dTTP, 70 μM of DIG-11-dUTP, 1x PCR buffer, and 2.625 units of Enzyme mix, Expand High Fidelity (#1732641, Roche). The PCR conditions were set as follows: preheating at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 40 s and then final extension at 72°C for 7 min.
UV-crosslinked membranes were prehybridized with 10 ml of DIG EasyHyb for 30 min at 50°C. Thereafter, DIG-labelled DNA probes were hybridized overnight at 50°C in 15 ml of DIG EasyHyb. Membranes were washed every 15 min in 5×Saline Sodium Citrate (SSC)/0.1% SDS, 1×SSC/0.1% SDS and 0.5× SSC/0.1% SDS buffers at 50°C followed by washing with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween-20 pH 7.5) for 5 min at 37°C. Thereafter, membranes were blocked with blocking solution (#11585762001, Roche) for 45 min at 37°C, and probed with 75 mU/ml Anti-Digoxigenin-AP (#11093274001, Roche) in blocking solution for 45 min at 37°C. Membranes were then washed again in maleic acid wash buffer in two 15-min steps and equilibrated with detection buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5). Signals were visualized by CDP-star (#12041677001, Roche) with Fusion Fx Imaging System (Vilber-Lourmat).
Western blotting
One-hundred bacterial cultures at OD600 = 2.0 were centrifuged at 10,000 rpm for 2 min at 22°C and resuspended in 100 μl 1xProtein loading buffer, followed by incubation for 5 min at 95°C. Aliquots of 5 μl were separated on 10% TGX gel (Bio-Rad) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane using a Transblot Turbo Transfer System (Bio-Rad).
For the CLIP procedure, 5 μl aliquots of heat-denatured immunoprecipitated RNA-protein complex were loaded and separated via SDS-polyacrylamide gel electrophoresis (12% resolving gel) at 30 mA per gel while moving through the stacking gel, which was then increased to 40 mA. After electrophoresis, proteins were electro-transferred onto a PVDF membrane.
Transferred membranes were blocked in 1xTBS-T buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween20, pH 7.6) with 10% skim milk for 45 min. Thereafter, the membrane was probed overnight at 4°C with monoclonal α-FLAG (#31430, ThermoFisher Scientific; 1:10,000) antibody diluted in 1×TBS-T buffer containing 3% bovine serum albumin, washed thrice for 5 min each in 1×TBS-T buffer, probed for 1 h with anti-mouse-HRP-antibody (F1804, Sigma-Aldrich; 1:50,000) diluted in 1×TBS-T buffer containing 3% bovine serum albumin, and washed thrice for 5 min each in 1×TBS-T buffer. Chemiluminescent signals were detected using Immobilon Western Chemiluminescent HRP substrate (#WBKLS0500, Millipore) and measured with Fusion Fx Imaging System (Vilber-Lourmat).
Data availability
Raw sequence data are available in the DDBJ Sequenced Read Archive under the accession number DRA010307.
Table S1. RsmA/N peaks detected by CLIP-seq
Table S2. The list of strains, plasmids, and oligonucleotides
ACKNOWLEDGEMENTS
We thank Akiko Yokota and Tetsushi Suyama for their assistance with the registration of this study in AIST. We also thank Thorsten Bischler for his help with CLIP-seq analysis and Jörg Vogel for his helpful comments on this study.
This study was supported by Grant-in-Aid for JSPS Research Fellow Grant Number 17J10663. The sequencing of CLIP-seq libraries was supported by the JSPS KAKENHI Grant Number 16H06279 (PAGS).
K.C. designed study, performed experiments and data analysis, acquired funding, and wrote manuscript. L.B. performed data analysis and participated in manuscript writing. K.T. performed experiments. N.N and S.T. participated in manuscript writing and supervision.
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