In vitro-Charakterisierung regulatorischer und sensorischer RNAs in Prokaryoten

Im Rahmen dieser Arbeit wurden eine cis-kodierte und eine trans-kodierte Antisense-RNA aus Bacillus subtilis sowie eine RNA-Thermometerstruktur aus Salmonella enterica in vitro strukturell und funktionell charakterisiert. In vitro-Analysen zur inhibitorischen Wirkung der cis-kodierten Antisense-RNA RNAIII haben gezeigt, dass die Ausbildung einer vollständigen Duplex mit ihrer Target-RNA (RNAII) nicht erforderlich ist. Ein Bindungsintermediat, das lediglich die simultane Beteiligung der Stem-Loops L3 und L4 von RNAIII erfordert, reicht als inhibitorischer Komplex für die Regulation der Replikation des Streptokokkenplasmides pIP501 aus. SR1 aus Bacillus subtilis ist eine 205 nt lange trans-kodierte Antisense-RNA, die durch direkte Basenpaarung regulierend auf ihr Target, ahrC-mRNA, einwirken kann. SR1 induziert strukturelle Veränderungen stromabwärts der Ribosomenbindungsstelle von ahrC-mRNA, wodurch die Translationsinitiation von AhrC verhindert wird. Die Translationskontrolle des agsA-Transkriptes in Salmonella enterica erfolgt über eine RNA-Thermometer-Struktur, die keinem der bisher identifizierten RNA-Thermometertypen entspricht

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

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

Sim1 Is a Novel Regulator in the Differentiation of Mouse Dorsal Raphe Serotonergic Neurons

BACKGROUND: Mesencephalic dopaminergic neurons (mDA) and serotonergic (5-HT) neurons are clinically important ventral neuronal populations. Degeneration of mDA is associated with Parkinson's disease; defects in the serotonergic system are related to depression, obsessive-compulsive disorder, and schizophrenia. Although these neuronal subpopulations reveal positional and developmental relationships, the developmental cascades that govern specification and differentiation of mDA or 5-HT neurons reveal missing determinants and are not yet understood. METHODOLOGY: We investigated the impact of the transcription factor Sim1 in the differentiation of mDA and rostral 5-HT neurons in vivo using Sim1-/- mouse embryos and newborn pups, and in vitro by gain- and loss-of-function approaches. PRINCIPAL FINDINGS: We show a selective significant reduction in the number of dorsal raphe nucleus (DRN) 5-HT neurons in Sim1-/- newborn mice. In contrast, 5-HT neurons of other raphe nuclei as well as dopaminergic neurons were not affected. Analysis of the underlying molecular mechanism revealed that tryptophan hydroxylase 2 (Tph2) and the transcription factor Pet1 are regulated by Sim1. Moreover, the transcription factor Lhx8 and the modulator of 5-HT(1A)-mediated neurotransmitter release, Rgs4, exhibit significant higher expression in ventral hindbrain, compared to midbrain and are target genes of Sim1. CONCLUSIONS: The results demonstrate for the first time a selective transcription factor dependence of the 5-HT cell groups, and introduce Sim1 as a regulator of DRN specification acting upstream of Pet1 and Tph2. Moreover, Sim1 may act to modulate serotonin release via regulating RGS4. Our study underscores that subpopulations of a common neurotransmitter phenotype use distinct combinations of transcription factors to control the expression of shared properties

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 rdp1Δ 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 tRNATyr 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 tRNATyr 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 tRNATyr gene is the causative mutation for SAN