RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles.

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.

Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes.

The histone acetyltransferase (HAT) subunit of coactivator complex SAGA, Gcn5, stimulates eviction of promoter nucleosomes at certain highly expressed yeast genes, including those activated by transcription factor Gcn4 in amino acid-deprived cells; however, the importance of other HAT complexes in this process was poorly understood. Analyzing mutations that disrupt the integrity or activity of HAT complexes NuA4 or NuA3, or HAT Rtt109, revealed that only NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes and stimulating transcription of starvation-induced genes. NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, TBP recruitment, and transcription at most other genes expressed constitutively. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the most highly expressed subset including ribosomal protein genes, where Gcn5 contributes strongly to PIC assembly and transcription. Both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that might be feedback controlled by their HAT activities. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria.

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3' untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3' UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

Organ-specific effects on target binding due to knockout of thyroid hormone receptor α during Xenopus metamorphosis.

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRß. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thratmshi ) tadpoles, we have previously shown that TRα is important for T3-dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild-type and Xtr·thratmshi tails with or without T3 treatment by using chromatin immunoprecipitation-sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild-type but not Xtr·thratmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating T3-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.

eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide.

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

Design, synthesis, and anticancer evaluation of ammosamide B with pyrroloquinoline derivatives as novel BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4), which is a member of the bromodomain and extra-terminal domain (BET) family, plays an important role in the regulation of gene expression as the "reader" of epigenetic regulation. BRD4 has become a promising target to treat cancer, because the up-regulation of BRD4 expression is closely associated with the occurrence and development of various cancers. At present, several BRD4 inhibitors are in clinical trials for cancer therapy, but no BRD4 inhibitors are on the market. Here, we designed and synthesized a series of compounds bearing pyrrolo[4,3,2-de]quinolin-2(1H)-one scaffold through structural modification of natural products ammosamide B, which is a natural pyrroloquinoline derivative reported for its potential antitumor activity. All target compounds were evaluated for their BRD4 BD1 inhibition activities via the protein thermal shift assays or AlphaSceen assay. The representative compound 49 showed potent activity (IC50 = 120 nM). The co-crystal of compound 49 with BRD4 BD1 was solved to study the structure activity relationship, which showed that 49 could combine with the acetyl lysine binding site and formed a hydrogen bond with the conserved residue Asn140. The results demonstrate that compound 49 is worthy of further investigation as a promising BRD4 inhibitor.

Correction: Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

[This corrects the article DOI: 10.1371/journal.pgen.1007806.].

Thyroid Hormone Receptor α Controls the Hind Limb Metamorphosis by Regulating Cell Proliferation and Wnt Signaling Pathways in Xenopus tropicalis.

Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.

Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

Comprehensive RNA-Seq analysis of notochord-enriched genes induced during Xenopus tropicalis tail resorption.

Anuran metamorphosis is perhaps the most dramatic developmental process regulated by thyroid hormone (TH). One of the unique processes that occur during metamorphosis is the complete resorption of the tail, including the notochord. Interestingly, recent gene knockout studies have shown that of the two known vertebrate TH receptors, TRα and TRß, TRß appears to be critical for notochord regression during tail resorption in Xenopus tropicalis. To determine the mechanisms underlying notochord regression, we carried out a comprehensive gene expression analysis in the notochord during metamorphosis by using RNA-Seq analyses of whole tail at stage 60 before any noticeable tail length reduction, whole tail at stage 63 when the tail length is reduced by about one half, and the rest of the tail at stage 63 after removing the notochord. This allowed us to identify many notochord-enriched, metamorphosis-induced genes at stage 63. Future studies on these genes should help to determine if they are regulated by TRß and play any roles in notochord regression.

Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae.

BACKGROUND: Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. RESULTS: Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. CONCLUSIONS: Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Cadexomer iodine effectively reduces bacterial biofilm in porcine wounds ex vivo and in vivo.

Biofilms are prevalent in non-healing chronic wounds and implicated in delayed healing. Tolerance to antimicrobial treatments and the host's immune system leave clinicians with limited interventions against biofilm populations. It is therefore essential that effective treatments be rigorously tested and demonstrate an impact on biofilm across multiple experimental models to guide clinical investigations and protocols. Cadexomer iodine has previously been shown to be effective against biofilm in various in vitro models, against methicillin-resistant Staphylococcus aureus biofilm in mouse wounds, and clinically in diabetic foot ulcers complicated by biofilm. Similarities between porcine and human skin make the pig a favoured model for cutaneous wound studies. Two antiseptic dressings and a gauze control were assessed against mature biofilm grown on ex vivo pig skin and in a pig wound model. Significant reductions in biofilm were observed following treatment with cadexomer iodine across both biofilm models. In contrast, silver carboxymethylcellulose dressings had minimal impact on biofilm in the models, with similar results to the control in the ex vivo model. Microscopy and histopathology indicate that the depth of organisms in wound tissue may impact treatment effectiveness. Further work on the promising biofilm efficacy of cadexomer iodine is needed to determine optimal treatment durations against biofilm.

caOmicsV: an R package for visualizing multidimensional cancer genomic data.

BACKGROUND: Translational genomics research in cancers, e.g., International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), has generated large multidimensional datasets from high-throughput technologies. Data analysis at multidimensional level will greatly benefit clinical applications of genomic information in diagnosis, prognosis and therapeutics of cancers. To help, tools to effectively visualize integrated multidimensional data are important for understanding and describing the relationship between genomic variations and cancers. RESULTS: We implemented the R package, caOmicsV, to provide methods under R environment to visualize multidimensional cancer genomic data in two layouts: matrix layout and combined biological network and circular layout. Both layouts support to display sample information, gene expression (e.g., RNA and miRNA), DNA methylation, DNA copy number variations, and summarized data. A set of supplemental functions are included in the caOmicsV package to help users in generation of plot data sets from multiple genomic datasets with given gene names and sample names. Default plot methods for both layouts for easy use are also implemented. CONCLUSION: caOmicsV package provides an easy and flexible way to visualize integrated multidimensional cancer genomic data under R environment.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Amino acid transporter SLC7A5 regulates cell proliferation and secretary cell differentiation and distribution in the mouse intestine.

The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.

RCircos: an R package for Circos 2D track plots.

BACKGROUND: Circos is a Perl language based software package for visualizing similarities and differences of genome structure and positional relationships between genomic intervals. Running Circos requires extra data processing procedures to prepare plot data files and configure files from datasets, which limits its capability of integrating directly with other software tools such as R. Recently published R Bioconductor package ggbio provides a function to display genomic data in circular layout based on multiple other packages, which increases its complexity of usage and decreased the flexibility in integrating with other R pipelines. RESULTS: We implemented an R package, RCircos, using only R packages that come with R base installation. The package supports Circos 2D data track plots such as scatter, line, histogram, heatmap, tile, connectors, links, and text labels. Each plot is implemented with a specific function and input data for all functions are data frames which can be objects read from text files or generated with other R pipelines. CONCLUSION: RCircos package provides a simple and flexible way to make Circos 2D track plots with R and could be easily integrated into other R data processing and graphic manipulation pipelines for presenting large-scale multi-sample genomic research data. It can also serve as a base tool to generate complex Circos images.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Liver development during Xenopus tropicalis metamorphosis is controlled by T3-activation of WNT signaling.

Thyroid hormone (T3) regulates vertebrate organ development, growth, and metabolism through the T3 receptor (TR). Due to maternal influence in mammals, it has been difficult to study if and how T3 regulates liver development. Liver remodeling during anuran metamorphosis resembles liver maturation in mammals and is controlled by T3. We generated Xenopus tropicalis animals with both TRα and TRß genes knocked out and found that TR double knockout liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate urea cycle gene expression. RNA-seq analysis showed that T3 activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely promoted the proliferation and maturation of hepatocytes. Our study offers new insights into not only how T3 regulates liver development but also on potential means to improve liver regeneration.

Comparative Analysis of Transcriptome Profiles Reveals Distinct and Organ-Dependent Genomic and Nongenomic Actions of Thyroid Hormone in Xenopus tropicalis Tadpoles.

Background: Thyroid hormone (triiodothyronine [T3]) is essential for development and organ metabolism in all vertebrates. T3 has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via T3 receptors (TRs) in vertebrate development, mostly through TR-knockout and TR-knockin studies, little is known about the effects of T3 on gene expression in animals in the absence of TR. We have been studying Xenopus metamorphosis as a model for mammalian postembryonic development, a period around birth when plasma T3 level peaks and many organs/tissues mature into their adult forms. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals. This offers an opportunity to compare the effects of T3 on global gene expression in tadpole tissues in the presence or absence of TR. Methods: We analyzed the effects of T3 on gene expression in tadpole tail and intestine by using RNA-seq analysis on wild-type and TRDKO tadpoles with or without T3 treatment. Results: We observed that removing TRs reduced the number of genes regulated by T3 in both organs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that T3 affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms and KEGG pathways that were enriched among genes regulated in wild-type tissues are likely involved in mediating the effects of T3 on metamorphosis, for example, those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among T3-regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to "metabolism" and "immune response" were highly enriched among T3-regulated genes. We further observed strong divergence in the TR-independent nongenomic effects of T3 in the intestine and tail. Conclusions: Our data suggest that T3 has distinct and organ-dependent effects on gene expression in developing tadpoles. The TR-mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of T3 on metamorphosis. T3 appears to have a major effect on metabolism and immune response via TR-independent nongenomic processes.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.