Regulation of alternative splicing and polyadenylation in neurons.

Cell-type-specific gene expression is a fundamental feature of multicellular organisms and is achieved by combinations of regulatory strategies. Although cell-restricted transcription is perhaps the most widely studied mechanism, co-transcriptional and post-transcriptional processes are also central to the spatiotemporal control of gene functions. One general category of expression control involves the generation of multiple transcript isoforms from an individual gene, whose balance and cell specificity are frequently tightly regulated via diverse strategies. The nervous system makes particularly extensive use of cell-specific isoforms, specializing the neural function of genes that are expressed more broadly. Here, we review regulatory strategies and RNA-binding proteins that direct neural-specific isoform processing. These include various classes of alternative splicing and alternative polyadenylation events, both of which broadly diversify the neural transcriptome. Importantly, global alterations of splicing and alternative polyadenylation are characteristic of many neural pathologies, and recent genetic studies demonstrate how misregulation of individual neural isoforms can directly cause mutant phenotypes.

Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

Post-Transcriptional Regulation of RseA by Small RNAs RyhB and FnrS in Escherichia coli.

RseA is the critical central regulator of the σE-dependent stress response in E. coli and other related bacteria. The synthesis of RseA is controlled at the transcriptional level by several promoters and transcriptional regulators, including σE itself at two σE-dependent promoters: rpoE P and rseA P3. The presence of these two independent polycistrons encoding rseA is potentially redundant. We hypothesized that post-transcriptional control of the rseA P3 transcript was necessary to overcome this redundancy. However, to date, nothing is known about the post-transcriptional control of the rseA P3 transcript. We executed a targeted genetic screen to identify small RNA regulators of the rseA P3 transcript and identified RyhB and FnrS as small RNA activators of the RseA P3 transcript. Through genetic analysis, we confirmed that a direct interaction occurs between RyhB and RseA. We also identified sequences within the 5' untranslated region (UTR) of RseA that were inhibitory for RseA expression. Point mutations predicted to prevent an interaction between RyhB and RseA resulted in increased RseA expression. Taken together, this suggests that the 5' UTR of the RseAP3 transcript prevents optimal expression of RseA, preventing redundancy due to RseA expression from the σE-dependent rpoE P, and this is overcome by the stimulatory activity of RyhB and FnrS.

Ketamine interactions with gut-microbiota in rats: relevance to its antidepressant and anti-inflammatory properties.

BACKGROUND: Appreciable evidence suggest that dysbiosis in microbiota, reflected in gut microbial imbalance plays a key role in the pathogenesis of neuropsychiatric disorders including depression and inflammatory diseases. Recently, the antidepressant properties of ketamine have gained prominence due to its fast and long lasting effects. Additional uses for ketamine in inflammatory disorders such as irritable bowel syndrome have been suggested. However, ketamine's exact mechanism of action and potential effects on microbiome is not known. Here, we examined the effects of low dose ketamine, known to induce antidepressant effects, on stool microbiome profile in adult male Wistar rats. Animals (5/group) were injected intraperitoneally with ketamine (2.5 mg/kg) or saline, daily for 7 days and sacrificed on day 8 when intestinal stools were collected and stored at - 80 °C. DNA was extracted from the samples and the 16 S rRNA gene-based microbiota analysis was performed using 16S Metagenomics application. RESULTS: At genus-level, ketamine strikingly amplified Lactobacillus, Turicibacter and Sarcina by 3.3, 26 and 42 fold, respectively. Conversely, opportunistic pathogens Mucispirillum and Ruminococcus were reduced by approximately 2.6 and 26 fold, respectively, in ketamine group. Low levels of Lactobacillus and Turicibacter are associated with various disorders including depression and administration of certain species of Lactobacillus ameliorates depressive-like behavior in animal models. Hence, some of the antidepressant effects of ketamine might be mediated through its interaction with these gut bacteria. Additionally, high level of Ruminococcus is positively associated with the severity of irritable bowel syndrome (IBS), and some species of Mucispirillum have been associated with intestinal inflammation. Indirect evidence of anti-inflammatory role of Sarcina has been documented. Hence, some of the anti-inflammatory effects of ketamine and its usefulness in specific inflammatory diseases including IBS may be mediated through its interaction with these latter bacteria. CONCLUSION: Our data suggest that at least some of the antidepressant and anti-inflammatory effects of daily ketamine treatment for 7 days may be mediated via its interaction with specific gut bacteria. These findings further validate the usefulness of microbiome as a target for therapeutic intervention and call for more detailed investigation of microbiome interaction with central mediators of mood and/or inflammatory disorders.

An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis.

TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli.

Previous work demonstrated that efficient RNA Polymerase sigma S-subunit (RpoS) translation requires the N6-isopentenyladenosine i6A37 transfer RNA (tRNA) modification for UUX-Leu decoding. Here we investigate the effect of two additional tRNA modification systems on RpoS translation; the analysis was also extended to another High UUX-leucine codon (HULC) protein, Host Factor for phage Qß (Hfq). One tRNA modification, the addition of the 2'-O-methylcytidine/uridine 34 (C/U34m) tRNA modification by tRNA (cytidine/uridine-2'O)-ribose methyltransferase L (TrmL), requires the presence of the N6-isopentenyladenosine 37 (i6A37) and therefore it seemed possible that the defect in RpoS translation in the absence of i6A37 prenyl transferase (MiaA) was in fact due to the inability to add the C/U34m modification to UUX-Leu tRNAs. The second modification, addition of 2-thiouridine (s²U), part of (mnm5s²U34), is dependent on tRNA 2-thiouridine synthesizing protein A (TusA), previously shown to affect RpoS levels. We compared expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons were changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolished PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppressed the trmL requirement for PBAD-rpoS990-lacZ expression. Thus, it is likely that the C/U34m and s²U34 tRNA modifications are necessary for full rpoS translation. We also measured PBAD-hfq306-lacZ translational fusion activity in the absence of C/U34m (trmL) or i6A37 (miaA). The absence of i6A37 resulted in decreased PBAD-hfq306-lacZ expression, consistent with a role for i6A37 tRNA modification for hfq translation.

The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation.

The translation of rpoS(σ(S)), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in theN(6)-isopentyl-2-thiomethyl adenosinemethyl adenosine 37 (ms(2)i(6)A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraPas another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. AniraP-lacZ fusion was significantly decreased in the abse nce of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured ß-galactosidase-specific activity of miaA(-)rpo Sandira P translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and notira P, via overexpression of tRNA(LeuX)but not tRNA(LeuZ) We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification.