The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition.

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.

Investigating RNA-Protein Interactions in Neisseria meningitidis by RIP-Seq Analysis.

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.

The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

Neisseria meningitidis, a commensal ß-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.

The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

Investigating CRISPR RNA Biogenesis and Function Using RNA-seq.

The development of deep sequencing technology has greatly facilitated transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacteria. Moreover, RNA-seq is nowadays widely used for gene expression profiling and about to replace hybridization-based approaches such as microarrays. RNA-seq has also informed about the biogenesis and function of CRISPR RNAs (crRNAs) of different types of bacterial RNA-based CRISPR-Cas immune systems. Here we describe several studies that employed RNA-seq for crRNA analyses, with a particular focus on a differential RNA-seq (dRNA-seq) approach, which can distinguish between primary and processed transcripts and allows for a genome-wide annotation of transcriptional start sites. This approach helped to identify a new crRNA biogenesis pathway of Type II CRISPR-Cas systems that involves a trans-encoded small RNA, tracrRNA, and the host factor RNase III.

Same same but different: new structural insight into CRISPR-Cas complexes.

Three papers in this issue of Molecular Cell report on the structure and functional activity of type III CRISPR-Cas effector complexes, revealing novel and conserved features of the ribonucleoprotein particles that underlie prokaryotic genome defense. The new structures suggest that type I and type III complexes follow the same architectural principles and are most likely descendants of a common ancestor, the differences in RNA and protein sequences and structure of individual components notwithstanding.

CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation.

A Cas protein from the CRISPR defence system against foreign DNA, also functions in endogenous gene regulation. Sampson et al (2013) have revealed that in pathogenic Francisella bacteria, the Cas9 protein guided by small RNAs represses the mRNA of a lipoprotein. This novel mechanism of post-transcriptional regulation enables the infecting bacteria to evade the TLR2-based innate immune response of its host. Thus, reminiscent of eukaryotic RNAi where some proteins facilitate both genome defence and gene regulation, a central prokaryotic RNAi protein not only destroys invading DNA but also controls mRNA expression.

High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates.

Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA-seq (dRNA-seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA-seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA-seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA-seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.

Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis.

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5' ends formed by transcription and not by processing. Although crRNA 3' end formation involves RNase III and trans-encoded tracrRNA, as in other type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/Cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.

Two CRISPR-Cas systems in Methanosarcina mazei strain Gö1 display common processing features despite belonging to different types I and III.

The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5' repeat tags. The 3'-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3'-tag). The analysis further revealed a 5'-hydroxy and 3'-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.

H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO.

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

A dual-function sRNA from B. subtilis: SR1 acts as a peptide encoding mRNA on the gapA operon.

Small non-coding RNAs (sRNAs) have been found to regulate gene expression in all three kingdoms of life. So far, relatively little is known about sRNAs from Gram-positive bacteria. SR1 is a regulatory sRNA from the Bacillus subtilis chromosome that inhibits by base-pairing translation initiation of ahrC mRNA encoding a transcriptional activator of the arginine catabolic operons. Here we present a novel target of SR1, the glycolytic gapA operon. Both microarray and Northern blot analyses show that the amount of gapA operon mRNA is significantly higher in the presence of SR1 when cells were grown in complex medium until stationary phase. Translational lacZ fusions and toeprinting analyses demonstrate that SR1 does not promote translation of gapA mRNA. By contrast, the half-life of gapA operon mRNA is strongly reduced in the sr1 knockout strain. SR1 does not act as a base-pairing sRNA on gapA operon mRNA. Instead, we demonstrate that the 39 aa peptide encoded by SR1, SR1P, is responsible for the effect of SR1 on the gapA operon. We show that SR1P binds GapA, thereby stabilizing the gapA operon mRNA by a hitherto unknown mechanism. SR1 is the first dual-function sRNA found in B. subtilis.

Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA.

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5'-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Delta cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.

Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene.

Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.

CodY activates transcription of a small RNA in Bacillus subtilis.

Regulatory small RNAs (sRNAs) in bacterial genomes have become a focus of research over the past 8 years. Whereas more than 100 such sRNAs have been found in Escherichia coli, relatively little is known about sRNAs in gram-positive bacteria. Using a computational approach, we identified two sRNAs in intergenic regions of the Bacillus subtilis genome, SR1 and SR2 (renamed BsrF). Recently, we demonstrated that SR1 inhibits the translation initiation of the transcriptional activator AhrC. Here, we describe detection of BsrF, its expression profile, and its regulation by CodY. Furthermore, we mapped the secondary structure of BsrF. BsrF is expressed in complex and minimal media in all growth phases in B. subtilis and, with a similar expression profile, also in Bacillus amyloliquefaciens. Neither overexpression nor deletion of bsrF affected the growth of B. subtilis. BsrF was found to be long-lived in complex and minimal media. Analysis of 13 putative transcription factor binding sites upstream of bsrF revealed only an effect for CodY. Here, we showed by using Northern blotting, lacZ reporter gene fusions, in vitro transcription, and DNase I footprinting that the transcription of bsrF is activated by CodY in the presence of branched-chain amino acids and GTP. Furthermore, BsrF transcription was increased 1.5- to 2-fold by glucose in the presence of branched-chain amino acids, and this increase was independent of the known glucose-dependent regulators. BsrF is the second target for which transcriptional activation by CodY has been discovered.

FourU: a novel type of RNA thermometer in Salmonella.

The translation of many heat shock and virulence genes is controlled by RNA thermometers. Usually, they are located in the 5'-untranslated region (5'-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Destabilization of the structure at elevated temperature permits ribosome binding and translation initiation. We have identified a new type of RNA thermometer in the 5'-UTR of the Salmonella agsA gene, which codes for a small heat shock protein. Transcription of the agsA gene is controlled by the alternative sigma factor sigma(32). Additional translational control depends on a stretch of four uridines that pair with the SD sequence. Mutations in this region affect translation in vivo. Structure probing experiments demonstrate a temperature-controlled opening of the SD region in vitro. Toeprinting (primer extension inhibition) shows that ribosome binding is dependent on high temperatures. Together with a postulated RNA thermometer upstream of the Yersinia pestis virulence gene lcrF (virF), the 5'-UTR of Salmonella agsA might be the founding member of a new class of RNA thermometers that we propose to name 'fourU' thermometers.

In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA.

Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5' part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

Antisense RNA-mediated transcriptional attenuation in plasmid pIP501: the simultaneous interaction between two complementary loop pairs is required for efficient inhibition by the antisense RNA.

Streptococcal plasmid pIP501 uses antisense RNA-mediated transcriptional attenuation to regulate its replication. Previous in vitro assays suggested that binding intermediates between RNAII (sense RNA) and RNAIII (antisense RNA) are sufficient for inhibition, and a U-turn structure on RNAII loop L1 was found to be crucial for the interaction with RNAIII. Here, sequence and structural requirements for an efficient RNAII-RNAIII interaction were investigated. A detailed probing of RNA secondary structure combined with in vitro single-round transcription assays indicated that complex formation between the two molecules progresses into the lower stems of both loop pairs of the sense and antisense RNAs, but that the complex between RNAII and RNAIII is not a full duplex. Stem-loops L3 and L4 were required to be linked to one other for efficient contact with the complementary loops L2 and L1 of the sense RNA, indicating a simultaneous interaction between these two loop pairs. Thereby, the sequence and length of the spacer connecting L3 and L4 were shown not to be important for inhibition.

The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism.

Whereas about 70 small non-coding RNAs have been found in the Escherichia coli genome, relatively little is known about regulatory RNAs from Gram-positive bacteria. Here, we demonstrate that the recently identified small untranslated RNA SR1 from the Bacillus subtilis genome is a regulatory RNA involved in fine-tuning of arginine catabolism. 2D protein gel electrophoresis indicated three possible SR1 targets that are regulated by the transcriptional activator AhrC, which was shown to be the primary target of SR1. In vitro pairing studies and an in vivo reporter gene test demonstrated a specific interaction between SR1 and ahrC mRNA. This interaction did not lead to degradation of ahrC mRNA, but inhibited translation at a post-initiation stage. Our data show that the Hfq chaperone was not required for the stabilization of SR1 in vivo. The amount of SR1 was increased upon addition of l-arginine and l-ornithine, but not l-citrulline or l-proline.