Ramakanth Madhugiri

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5‐insertion in the RNase J‐encoding gene was compared to the wild‐type and a difference in the length of the 5.8S‐like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′‐end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.

Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs

Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40–150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its target rpoS in E. coli is growth phase dependent.

OxyS is one of at least three small non-coding RNAs, which affect rpoS expression. It is induced under oxidative stress and reduces the levels of the stationary phase sigma factor RpoS. We analyzed the turn-over of OxyS and rpoS mRNA in early exponential and in stationary growth phase in different E. coli strains to learn more about the mechanisms of processing and about a possible impact of processing on growth-dependent regulation. We could not attribute a major role of RNase E, RNase III, PNPase or RNase II on OxyS turn-over in exponential growth phase. Only the simultaneous lack of RNase E, PNPase and RNase II activity resulted in some stabilization of OxyS in exponential growth phase, implying the action of multiple ribonucleases on OxyS turn-over. A major role of RNase E on OxyS stability was observed in stationary phase and was dependent on the presence of the RNA binding protein Hfq and of DsrA, one of the other small RNAs binding to rpoS mRNA. Our data also confirm a role of RNase III in rpoS turn-over, however, only in exponential growth phase. We conclude that OxyS and rpoS mRNA processing is influenced by different RNases and additional factors like Hfq and DsrA and that the impact of these factors is strongly dependent on growth phase.

Turn-over of the small non-coding RNA RprA in E. coli is influenced by osmolarity

The sRNA RprA is known to activate rpoS translation in E. coli in an osmolarity-dependent manner. We asked whether RprA stability contributes to osmolarity-dependent regulation and how the RNA binding protein Hfq and the major E. coli endonucleases contribute to this turn-over. The study reveals that osmolarity-dependent turn-over of RprA indeed contributes to its osmolarity-dependent abundance. RprA is stabilized by the RNA chaperone Hfq and in absence of Hfq its turn-over is no longer osmolarity-dependent. The stability of the RprA target mRNA rpoS shows a lower extent of osmolarity dependence, which differs from the profile observed for RprA. Thus, the effect of sucrose is specific for individual RNAs. We can attribute a role of the endoribonuclease RNase E in turn-over of RprA and an indirect effect of the endoribonuclease III in vivo. In addition, RprA is stabilized by the presence of rpoS suggesting that hybrid formation with its target may protect it against ribonucleases. In vitro RprA is cleaved by the RNase E containing degradosome and by RNase III and rpoS interferes with RNase III cleavage. We also show that temperature affects the stabilities of the sRNAs binding to rpoS and of rpoS mRNA itself differentially and that higher stability of DsrA with decreasing temperature may contribute to its high abundance at lower temperatures. This study demonstrates that environmental parameters can affect the stability of sRNAs and consequently their abundance.

RNA structure analysis of alphacoronavirus terminal genome regions

Abstract Coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. Interactions of the viral replicase complex with cis-acting RNA elements located in the 5′ and 3′-terminal genome regions ensure the specific replication of viral RNA. Over the past years, boundaries and structures of cis-acting RNA elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. Here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and RNA structure probing studies to identify and characterize putative cis-acting RNA elements in alphacoronaviruses. The study suggests significant RNA structure conservation among members of the genus Alphacoronavirus but also across genus boundaries. Overall, the conservation pattern identified for 5′ and 3′-terminal RNA structural elements in the genomes of alpha- and betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the Coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting RNA elements.

Broad-spectrum antiviral activity of the eIF4A inhibitor silvestrol against corona- and picornaviruses

Abstract Coronaviruses (CoV) and picornaviruses are plus‐strand RNA viruses that use 5′ cap‐dependent and cap‐independent strategies, respectively, for viral mRNA translation initiation. Here, we analyzed the effects of the plant compound silvestrol, a specific inhibitor of the DEAD‐box RNA helicase eIF4A, on viral translation using a dual luciferase assay and virus‐infected primary cells. Silvestrol was recently shown to have potent antiviral activity in Ebola virus‐infected human macrophages. We found that silvestrol is also a potent inhibitor of cap‐dependent viral mRNA translation in CoV‐infected human embryonic lung fibroblast (MRC‐5) cells. EC50 values of 1.3 nM and 3 nM silvestrol were determined for MERS‐CoV and HCoV‐229E, respectively. For the highly pathogenic MERS‐CoV, the potent antiviral activities of silvestrol were also confirmed using peripheral blood mononuclear cells (PBMCs) as a second type of human primary cells. Silvestrol strongly inhibits the expression of CoV structural and nonstructural proteins (N, nsp8) and the formation of viral replication/transcription complexes. Furthermore, potential antiviral effects against human rhinovirus (HRV) A1 and poliovirus type 1 (PV), representing different species in the genus Enterovirus (family Picornaviridae), were investigated. The two viruses employ an internal ribosomal entry site (IRES)‐mediated translation initiation mechanism. For PV, which is known to require the activity of eIF4A, an EC50 value of 20 nM silvestrol was determined in MRC‐5 cells. The higher EC50 value of 100 nM measured for HRV A1 indicates a less critical role of eIF4A activity in HRV A1 IRES‐mediated translation initiation. Taken together, the data reveal a broad‐spectrum antiviral activity of silvestrol in infected primary cells by inhibiting eIF4A‐dependent viral mRNA translation. Graphical abstract Figure. No caption available. HighlightsThe eIF4A inhibitor silvestrol is a potent antiviral compound that inhibits the replication of coronaviruses.Silvestrol is also effective against picornaviruses with an eIF4A‐dependent Type 1 IRES element.In primary cells silvestrol has potent antiviral activity and low toxicity.Targeting the host factor eIF4A is a promising broad‐spectrum antiviral strategy.

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti

The ribonucleases (RNases) E and J play major roles in E. coli and Bacillus subtilis, respectively, and co-exist in Sinorhizobium meliloti. We analysed S. meliloti 2011 mutants with mini-Tn5 insertions in the corresponding genes rne and rnj and found many overlapping effects. We observed similar changes in mRNA levels, including lower mRNA levels of the motility and chemotaxis related genes flaA, flgB and cheR and higher levels of ndvA (important for glucan export). The acyl-homoserine lactone (AHL) levels were also higher during exponential growth in both RNase mutants, despite no increase in the expression of the sinI AHL synthase gene. Furthermore, several RNAs from both mutants migrated aberrantly in denaturing gels at 300 V but not under stronger denaturing conditions at 1300 V. The similarities between the two mutants could be explained by increased levels of the key methyl donor S-adenosylmethionine (SAM), since this may result in faster AHL synthesis leading to higher AHL accumulation as well as in uncontrolled methylation of macromolecules including RNA, which may strengthen RNA secondary structures. Indeed, we found that in both mutants the N6-methyladenosine content was increased almost threefold and the SAM level was increased at least sevenfold. Complementation by induced ectopic expression of the respective RNase restored the AHL and SAM levels in each of the mutants. In summary, our data show that both RNase E and RNase J are needed for SAM homeostasis in S. meliloti.

Detection of Very Long Antisense Transcripts by Whole Transcriptome RNA-Seq Analysis of Listeria monocytogenes by Semiconductor Sequencing Technology

The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

Coronavirus cis-Acting RNA Elements.

Abstract Coronaviruses have exceptionally large RNA genomes of approximately 30 kilobases. Genome replication and transcription is mediated by a multisubunit protein complex comprised of more than a dozen virus-encoded proteins. The protein complex is thought to bind specific cis-acting RNA elements primarily located in the 5′- and 3′-terminal genome regions and upstream of the open reading frames located in the 3′-proximal one-third of the genome. Here, we review our current understanding of coronavirus cis-acting RNA elements, focusing on elements required for genome replication and packaging. Recent bioinformatic, biochemical, and genetic studies suggest a previously unknown level of conservation of cis-acting RNA structures among different coronavirus genera and, in some cases, even beyond genus boundaries. Also, there is increasing evidence to suggest that individual cis-acting elements may be part of higher-order RNA structures involving long-range and dynamic RNA–RNA interactions between RNA structural elements separated by thousands of nucleotides in the viral genome. We discuss the structural and functional features of these cis-acting RNA elements and their specific functions in coronavirus RNA synthesis.