Here, we identify a novel role of Hfq in ribosome biogenesis. Inactivation of Hfq leads to accumulation of 17S rRNA and reduced levels of 70S ribosomes in E. coli . Using in vivo and in vitro approaches, including ribosome profiling, we demonstrate that Hfq deletion affects the ribosome pool with direct effects on translation efficiency and fidelity. Our data propose Hfq as a novel auxiliary ribosome biogenesis factor. This expands the functional spectrum of this RNA chaperone beyond the sRNA‐biology with impact on rRNA processing, ribosome biogenesis, and translation fidelity.

Hfq interacts in vitro with the 16S rRNA (de Haseth & Uhlenbeck, 1980 ) although the functional role of this interaction has remained elusive. Furthermore, rRNA molecules are commonly found in Hfq‐enriched co‐immunoprecipitations, what is usually regarded as a background noise in transcriptomic studies (Zhang et al , 2003 ; Sittka et al , 2008 ; Bilusic et al , 2014 ). A cross‐linking‐based study in E. coli suggests interactions of Hfq with rRNA in vivo (Tree et al , 2014 ). An interaction between Hfq and S12 protein of the 30S ribosomal subunit has been previously reported, yet lacking mechanistic details on its role (Strader et al , 2013 ). Clearly, Hfq interacts with rRNA but is this a functional or redundant interaction?

The bacterial RNA‐binding protein Hfq is a member of the Sm/Lsm superfamily of proteins with homologues in all domains of life (Wilusz & Wilusz, 2013 ). Hfq is an RNA chaperone which facilitates basepairing between small regulatory RNAs (sRNAs) and their mRNA targets. Consequently, Hfq controls the expression of many mRNAs either positively or negatively (Vogel & Luisi, 2011 ; Hajnsdorf & Boni, 2012 ; Updegrove et al , 2016 ). Importantly, in many bacteria, Hfq is not required for the sRNA‐dependent pathways (Christiansen et al , 2006 ; Rochat et al , 2015 ), suggesting other yet undefined function(s) of Hfq beyond regulation of sRNA activity.

In prokaryotes, the small 30S ribosomal subunit contains 16S rRNA whereas 23S and 5S rRNA are the major components of the large 50S ribosomal subunit. The two asymmetric subunits include numerous r‐proteins and associate to form the functionally active 70S ribosome (Shajani et al , 2011 ). Many auxiliary ribosome biogenesis factors, including GTPases, rRNA modification enzymes, helicases, and other maturation factors, assist rRNA folding and r‐protein assembly pathway (Davis & Williamson, 2017 ). Strikingly, mutations affecting many of these accessory proteins cause dysfunctional ribosomes. In humans, such mutations were shown to lead to severe diseases, collectively referred to as ribosomopathies (Narla & Ebert, 2010 ).

Ribosomal RNA (rRNA) represents more than 80% of total RNA in the cell and along with a plethora of ribosomal proteins (r‐proteins) constitutes the ribosome—the biosynthetic machinery of the cell. Ribosome biogenesis is a multi‐step hierarchically ordered process in which processing of rRNA precursor (pre‐rRNA) is a critical step. Emerging evidence suggests that pre‐rRNA maturation serves as a quality control to guarantee the integrity of the functional ribosome. In Escherichia coli , RNase III is responsible for the initial cleavages that separate individual rRNA precursors, followed by subsequent 5′ and 3′ processing by multiple ribonucleases to generate the 16S, 23S, and 5S rRNAs necessary to assemble the mature ribosomal subunits (Deutscher, 2009 ). Alterations in pre‐rRNA processing cause conformational changes in the final rRNA and lead to aberrantly assembled immature ribosomal particles with largely compromised translational accuracy (Liiv & Remme, 2004 ; Roy‐Chaudhuri et al , 2010 ; Yang et al , 2014 ). A parallel can be drawn to eukaryotes in which rRNA maturation errors lead to the production of defective ribosomal subunits (Cole et al , 2009 ; Fujii et al , 2012 ; Karbstein, 2013 ).

Results

Hfq is required for 16S rRNA maturation The 16S rRNA arises from processing of the 17S rRNA precursor which harbors additional nucleotides (nts) at both extremities (Fig 1A). Hfq is a key regulator of cell physiology affecting gene expression in both exponential and stationary phases (Tsui et al, 1994; Muffler et al, 1997; De Lay et al, 2013). Thus, we compared the total RNA from wild‐type and Δhfq cells extracted from exponential and stationary phase cells by Northern blotting using specific probes complementary to 5′‐ or 3′‐ends of the 17S rRNA. In addition, we used probes corresponding to the internal regions of 16S rRNA or 23S rRNA (Fig 1B). Both 17S‐specific probes hybridized only to 17S rRNA, whereas the 16S‐probe identified both 16S and 17S rRNA. Notably, inactivation of Hfq in both growth phases resulted in higher levels of 17S rRNA with misprocessed extremities (28 and 148% increase in exponential and stationary phases, respectively), suggesting a role for Hfq in 16S rRNA maturation. On the other hand, Hfq did not significantly change the levels of 23S rRNA. The accumulation of 17S in the ∆hfq mutant compared to the wild‐type strain is observed over time in different points of the growth curve (Fig EV1A). Figure 1.Hfq is required for correct processing and folding of 16S rRNA Schematic representation of the RNase‐mediated processing of the 17S rRNA precursor into mature 16S rRNA. Northern blot analysis of total RNA extracted from cells in exponential (EXP) or stationary (STAT) growth phase. Samples were fractionated on a 4% polyacrylamide/7 M urea gel. A scheme of the probes binding to the rRNA sequence is displayed on the side. Electrophoretic mobility shift assays of Hfq binding to the 5′ and 3′ extremities of the 17S rRNA. Increasing amounts of Hfq hexamer were mixed with a constant amount of the specific 17S‐flanking sequences and resolved on a 6% (top panel) or 8% (bottom panel) native polyacrylamide gel. DMS and CMCT accessibility probing of the 16S rRNA. Reverse‐transcribed cDNA was fractionated on an 10% polyacrylamide/7 M urea gel. Residues with altered reactivities in the Δhfq mutant are indicated. The inset depicts the analyzed region of the 16S rRNA. Source data are available online for this figure. Source Data for Figure 1 [embj201797631-sup-0006-SDataFig1.pdf] Click here to expand this figure. Figure EV1.Hfq regulates 17S rRNA levels 17S rRNA accumulates over time in the Δhfq strain. Northern blot analysis of total RNA isolated at different timepoints following the growth curve of wild‐type and Δhfq cells. Samples were separated on a 4% polyacrylamide/7 M urea gel, and a probe specific for the 17S 5′‐end was used. Predicted Hfq‐binding motifs within the 17S rRNA flanking sequences. The Hfq‐binding motif (ARN)x is highlighted in blue. (R—purine; N—any nucleotide). Hfq preferentially binds to the (ARN)x motif in RNAs (Mikulecky et al, 2004; Link et al, 2009; Peng et al, 2014). Strikingly, both 5′ and 3′ extremities of the 17S rRNA carry several of these predicted Hfq‐binding sequences (Fig EV1B). To further assess the Hfq binding to these regions, we performed gel mobility shift experiments with constant amounts of the 5′‐end and 3′‐end sequences of 17S RNA and increasing levels of the Hfq protein (Fig 1C). Indeed, Hfq complexed with both 17S‐end sequences corroborating the idea that Hfq interacts in vitro with 17S rRNA specific sequences. The additional nucleotides from the 17S rRNA could interact with helix 1 and helix 2 of the mature 16S rRNA inducing alternative structures which would affect the folding of the central pseudoknot (Lodmell & Dahlberg, 1997; Roy‐Chaudhuri et al, 2010). To evaluate whether the deletion of Hfq leads to an altered conformation of the 16S rRNA, we performed RNA mapping using two chemical probes, dimethyl sulfate (DMS) and N‐cyclohexyl‐N′‐(2‐morpholinoethyl)carbodiimide (CMCT), in separate experiments (Fig 1D). A specific antisense primer to the 5′‐end of the 16S rRNA was used in the primer extension reactions which allowed good resolution of the 16S central pseudoknot that consists of helix 1 (nucleotides 9–13/21–25) and helix 2 (nucleotides 17–19/916–918; Brink et al, 1993). Several nucleotides accessible to DMS (adenosines and cytidines) or CMCT (uridines and guanosines) modification in the wild type were less reactive to these probes in the absence of Hfq (Fig 1D). Our data imply that the folding of the 16S rRNA is altered as consequence of Hfq inactivation, resulting in the structural occlusion of those residues. Altogether, our observations indicate that Hfq interacts with 17S rRNA and is necessary for the correct processing and folding of the mature 16S rRNA, and affects the formation of the central pseudoknot.

Inactivation of Hfq results in altered profiles of ribosome sedimentation Given that misprocessing of rRNA can be consequence of defects in ribosome assembly (Liiv & Remme, 2004; Roy‐Chaudhuri et al, 2010; Shajani et al, 2011), we next examined whether the defects in the maturation of 16S rRNA found in the Δhfq strain had consequences to the total amount of ribosomes. We profiled the ribosomes from exponential and stationary phase cultures of wild‐type and mutant Δhfq strains by sucrose gradient ultracentrifugation (Fig 2A and B). The ribosome identity in the different peaks was further confirmed by analyzing their rRNAs (Appendix Fig S1). In the wild‐type strain, under conditions that favor ribosome association (10 mM Mg2+), the peak corresponding to the small 30S subunits was nearly absent, while the amount of the 70S ribosomes was comparable between exponential and stationary phases (Fig 2A and B, and Appendix Fig S1). In clear contrast, the levels of the mature 70S ribosomes were reduced in the ∆hfq mutant as compared to the wild type, an effect particularly severe in the stationary phase. Additionally, free 30S accumulated in the ∆hfq, which again was more evident in stationary phase (Fig 2A and B). The complementation of the ∆hfq deletion in trans with a plasmid expressing Hfq (pHfq; Andrade et al, 2012) raised the amount of mature ribosomes to levels comparable to that of the wild‐type strain (Fig 2A and B, Appendix Fig S1). Strikingly, the plasmid expressing Hfq rescued the defects in the ribosomal amounts isolated from the Hfq deletion strain. Note that the Δhfq strain transformed with the empty vector was essentially identical to the Δhfq strain suggesting no effects of the transformation itself. Figure 2.Defective ribosome biogenesis in the Δhfq strain A, B. (Left panels) Ribosomes from cells in the exponential or stationary phase were fractionated on sucrose density gradients in 10 mM Mg 2+ to stabilize 70S particles with and without trans ‐complementation of hfq gene using pBAD24 plasmid. Ribosome species are identified over each peak; top and bottom denote the lowest (15%) and highest (45%) sucrose concentration in the gradient, respectively. (Right panels) Ribosomes purified from cells in exponential and stationary phases fractionated on sucrose density gradients at low 0.1 mM Mg 2+ concentration to promote 70S dissociation into free 30S and 50S subunits. Top and bottom denote the lowest (10%) and highest (30%) sucrose concentration in the gradient, respectively.

C. Serial dilutions (with 1:10 steps) of wild‐type and Δ hfq strains grown on LB‐agar plates at 37 or 16°C.

D. Comparison of mRNA expression (left) and protein production (right) of ribosomal proteins between wild‐type and Δhfq strains analyzed by RNA‐Seq (left) and ribosome profiling (right), respectively. Source data are available online for this figure. Source Data for Figure 2 [embj201797631-sup-0007-SDataFig2.pdf] Our data clearly demonstrate that the inactivation of Hfq leads to a reduction in the pool of 70S ribosomes in the cell. This could either result from imbalanced production of subunits or the occurrence of major defects in the assembly of the 70S particle upon inactivation of Hfq. To distinguish between these two possibilities, we further analyzed ribosomes under dissociative conditions (0.1 mM Mg2+) to guarantee that all ribosomal subunits would be in their free state. As observed in Fig 2A and B (right panels), both strains displayed comparable contents of 30S and 50S subunits irrespective of the growth phase. Hence, the lower levels of 70S ribosomes in the absence of Hfq (Fig 2A and B, left panels) are a consequence of defects in the 70S assembly. A well‐known hallmark of ribosome biogenesis defects in bacteria is the cold‐sensitive phenotype (Connolly & Culver, 2009). We next compared the growth of the wild‐type and Δhfq strains at 37 and 16°C (Fig 2C). Clearly, the Δhfq mutant exhibited the cold‐sensitive phenotype, with severe growth defects under cold shock but not at 37°C which correlated with the altered ribosome profile found in the absence of Hfq. This effect is reminiscent of the cold‐sensitive phenotype observed with different ribosome biogenesis factors like RbfA, KsgA, RimM, and RimO (Bylund et al, 1998; Connolly et al, 2008; Leong et al, 2013). rRNA synthesis feedforwards the synthesis of ribosomal proteins (Scott et al, 2014). Thus, to assess the expression of the r‐proteins, we used ribosome profiling which captures the positions of actively translating ribosomes and the ribosome‐protected fragments (RPFs) reporting on differences in gene expression at the level of translation (Ingolia et al, 2009; Li et al, 2014). This analysis was combined with RNA‐Seq to determine the mRNA expression levels and the regulation of gene expression at the level of transcription. Strikingly, all ribosomal proteins were significantly translationally downregulated in the Δhfq mutant strain while the levels of their transcripts remained unchanged or decreased to much lower extent (Figs 2D and EV2A, and Dataset EV1). Notably, among the significantly enriched gene ontology (GO) terms are genes participating in ribosome assembly (Dataset EV1). Furthermore, within the polycistronic mRNAs, the translation yields of the encoded r‐proteins differed (Fig EV2B and Dataset EV1) implying an independent translation initiation of the r‐proteins (Li et al, 2014). This expression pattern corroborates earlier observations for translational coupling of the expression of the ribosomal proteins and rRNA synthesis (Jinks‐Robertson & Nomura, 1981; Nomura, 1999). Cumulative profiles of all expressed genes do not differ between wild‐type and Δhfq strains, arguing against an effect of Hfq depletion on translation initiation (Fig EV3A). Click here to expand this figure. Figure EV2.Hfq affects the translation of ribosomal proteins Translational downregulation of ribosomal protein genes in the Δhfq strain. Comparison of mRNA expression (top panels) and protein production (bottom panels) of ribosomal proteins between the wild‐type and Δhfq strains used in this study and an additional wild‐type data set (WT#2; Hwang & Buskirk, 2017 Schematic organization of the ribosomal protein genes their respective operons. The operons were arbitrarily numbered 1–18. Click here to expand this figure. Figure EV3.Cumulative metagene profile and coverage profiles of selected downregulated genes in the wild‐type and Δhfq strain obtained by ribosome profiling Cumulative metagene profile of the read density as a function of position for RPFs. The expressed genes were individually normalized, aligned at the start codon, and averaged with equal weight. 1,075 and 1,231 genes from wild‐type and Δhfq strains, respectively, were considered in the analysis. Coverage profiles of selected downregulated genes representative of gene categories affected by Hfq deletion (Fig 4D). Elongation factor‐Ts (tsf), 23S rRNA 2′‐O‐ribose U2552 methyltransferase (rlmE) are included in the GO term “translation and ribosome” and threonine‐tRNA ligase (thrS) in the GO term “amino acid biosynthesis”. Coverage profiles of exemplified genes (bamA and rbsR) whose expression remained unchanged upon hfq deletion. BamA is an outer membrane protein assembly factor, and RbsR is a transcriptional factor of the operon involved in ribose catabolism and transport. Overall, our results show that the Hfq depletion leads to defects in ribosome biogenesis with consequences for the pool of mature 70S ribosomes and propose Hfq as an auxiliary factor which regulates ribosome biogenesis.

Hfq copurifies with immature 30S subunits We hypothesized that Hfq would preferably bind to immature 30S subunits as these can be enriched in 17S RNA. To test this, we purified immature 30S subunits from the knockout mutant of RbfA, a late assembly factor that accumulates pre‐30S particles enriched in 17S rRNA (Jones & Inouye, 1996; Bylund et al, 1998; Thurlow et al, 2016). Compared to the wild‐type, the ΔrbfA mutant showed a similar ribosome profile to the Δhfq mutant, with increasing levels 30S particles and lower levels 70S ribosomes (Fig 3A). The peak corresponding to the 30S fraction was recovered from the sucrose gradients of the ΔrbfA mutant, and the 30S subunits were purified in low salt conditions. In parallel, mature 30S subunits were obtained from dissociation of 70S ribosomes isolated from the wild type, also in low salt conditions. Purified 30S samples were then analyzed by mass spectrometry that identified proteins associated with 30S subunits. Most of the proteins identified corresponded to r‐proteins or known factors associated with ribosomes (Dataset EV2). Strikingly, Hfq was found to copurify only with immature 30S isolated from the ΔrbfA but not with the mature 30S isolated from the wild type (Fig 3B). The same 30S samples were analyzed by Western blotting using an anti‐Hfq antibody. Cell lysates of wild‐type and Δhfq strains and purified His‐tagged Hfq were used as controls. Western blot confirmed the presence of Hfq in the 30S purified from the ΔrbfA but not from the wild type, in total agreement with mass spectrometry data (Fig 3C). Overall, these results show that Hfq is copurifying with precursor 30S ribosomes in vivo and corroborates that Hfq is a novel factor that assists ribosome assembly. Figure 3.Hfq copurifies with immature 30S subunits Ribosomes from wild‐type and ΔrbfA exponential growing cells were analyzed on sucrose density gradients. The ΔrbfA mutant displays an altered ribosome profile with an increase in 30S and 50S subunits and a reduction of 70S ribosomes compared to the wild type. Representative proteins identified by mass spectrometry of purified 30S subunits from the wild‐type and ΔrbfA mutant. The measurement of all the peptides identified for each protein is shown as total ProtScore values calculated with the Pro Group™ Algorithm (Sciex), with a 95% confidence. The ratio between the ΔrbfA mutant and wild type are shown as normalized fold changes that are represented by positive or negative values corresponding to an increase or decrease, respectively, of the number of peptides found in the ΔrbfA mutant compared to the wild type. (ND, not detected). Western blot analysis of purified 30S subunits using an anti‐Hfq antibody. WT and Δhfq cell lysates as well as purified His‐Hfq protein were loaded as controls. Source data are available online for this figure. Source Data for Figure 3 [embj201797631-sup-0008-SDataFig3.pdf]

Hfq affects translation efficiency Altered ribosome biogenesis can lead to major defects in translation, and thus, we next assessed the translational status in the Δhfq mutant. Firstly, the Δhfq strain showed a reduced polysome fraction compared to that of the wild‐type strain (Fig 4A). Secondly, a global measurement of protein synthesis by pulse metabolic labeling confirmed a significant reduction of translation in Hfq‐depleted background (Fig 4B). Thirdly, the global translation efficiency, which was determined by the density of ribosomes from the ribosome profiling per mRNA from the RNA‐Seq dataset, was significantly reduced (Mann–Whitney U‐test or Wilcoxon rank‐sum test, P = 0.0001996; Fig 4C). Hence, the defects in rRNA precursor processing and ribosome biogenesis in the Δhfq mutant decreased translation volume and efficiency as compared to the parental strain. The well‐known importance of Hfq for stress response in E. coli could arise from this effect on translational capacity, and not only from Hfq's role in sRNA‐dependent regulation. Figure 4.The Δhfq strain displays reduced translation levels Polysomal fraction is reduced in Δhfq cells. Polysome profiles of the wild‐type and Δhfq strains were resolved on sucrose density gradient. Top and bottom denote the lowest (15%) and highest (50%) sucrose concentration in the gradient, respectively. In vivo incorporation of 35S‐methionine/cysteine translation assay in M9 medium. Data are normalized to the wild‐type strain and are means ± SEM (n = 3). ***P = 0.0004 (paired t‐test). Translation efficiency of wild‐type and Hfq‐depleted cells obtained by ribosome profiling. GO term analysis of translationally downregulated genes in the Δhfq. The top three affected categories are in bold. Full GO term analysis is included in Dataset EV1. We next asked whether these changes in translation efficiency are global or a fraction of genes escapes this trend. We performed a fold‐change analysis and ranked the genes according to the fold‐change in translation (i.e., only translationally up‐ or downregulated in the ribosome profiling set) but with unchanged mRNA expression from the RNA‐Seq experiment. Genes with changes in their RPF coverage higher than twofold were considered. The gene ontology (GO) analysis of the downregulated genes in Hfq‐depleted background showed several pathways being affected but with a significant GO term enrichment in genes participating in ribosome biogenesis, translation, and amino acid metabolism (Fig 4D and Dataset EV1). The complete list of genes with the GO categories is summarized in Dataset EV1. For comparison, density plots of representative examples downregulated in the Δhfq mutant (Fig EV3B) or with unaltered translation (Fig EV3C) are included. Notably, inactivation of Hfq augmented the mRNA levels of genes known to be regulated by Hfq‐dependent sRNAs, while their translation was only slightly affected (Dataset EV3).

Hfq affects translation fidelity The ribosomal tRNA accommodation site (A‐site) is formed by helix 44 of the 16S rRNA of the 30S subunit. Three aminoglycoside antibiotics, neomycin, paromomycin, and kanamycin, interact with the 16S rRNA near the A‐site and induce translational misreading (i.e., shift of the reading frame, stop‐codon readthrough; Foster & Champney, 2008). In the presence of sub‐lethal concentrations of neomycin, paromomycin, or kanamycin, the Δhfq mutant strain showed exacerbated growth defects relative to untreated Δhfq or wild‐type strains, suggesting that Hfq affects translation fidelity (Fig 5A). Additional aminoglycosides were further tested showing similar effect (Fig EV4). As control, the Δhfq strain did not show increased sensitivity to other classes of antibiotics, like colistin, which targets cell membrane (Figs 5A and EV4). We also investigated the misreading using a collection of widely used plasmids bearing lacZ as reporter (O'Connor et al, 1997). When compared with the isogenic parent, the Δhfq mutant showed a substantial increase in frameshifting, aberrant initiation from alternative start codon(s), and stop‐codon readthrough (Fig 5B), indicating that the accuracy of translation in Hfq‐depleted background is severely compromised. In sum, these data suggest that inactivation of Hfq decreases translation efficiency and enhances misreading of mRNA, implying a functional link between Hfq‐dependent alterations in rRNA processing, ribosome biogenesis, and translation fidelity. Figure 5.Hfq‐depleted cells exhibit increased codon misreading Serial dilutions (1:10) of wild‐type and Δhfq strains grown on LB‐agar plates at 37°C with and without sub‐lethal concentrations of neomycin (1 μg/ml), paromomycin (1 μg/ml), kanamycin (1 μg/ml), or colistin (0.1 μg/ml). Wild‐type and Δhfq strains expressing mutated lacZ gene (pSG plasmids) were tested for a frameshift mutation (+1 or −1), alternative initiation codons (CUG or AUA), or a non‐sense stop‐codon mutation (UGA or UAG). For each strain, the β‐galactosidase activity (in Miller units) was normalized to that of strain expressing the wild‐type lacZ. Data are means ± SEM (n = 3). **P < 0.01; *P < 0.02 (paired t‐test). Source data are available online for this figure. Source Data for Figure 5 [embj201797631-sup-0009-SDataFig5.pdf] Click here to expand this figure. Figure EV4.Additional Δhfq antibiotic sensitivity tested by serial dilution platting Serial dilutions (1:10) of wild‐type and Δhfq strains grown on LB‐agar plates at 37°C with and without sub‐lethal concentrations of gentamicin (0.1 μg/ml), streptomycin (1 μg/ml), cefotaxime (0.01 μg/ml), erythromycin (2 μg/ml), or ciprofloxacin (0.002 μg/ml).