Project description:In this study we have combined RNA-seq analysis of genome-wide transcriptional start sites with regular RNA-seq to study the transcriptional landscape of Mycobacterium tuberculosis during exponential culture and growth arrest using a starvation model where exponentially growing cells are incubated in PBS for 24 hours. Three independent biological replicates were used in this experiment.
Project description:Bacterial toxin-antitoxin systems help bacteria to reduce their metabolism in various stressful conditions and also play a part in generating antibiotic tolerant bacterial subpopulation called persisters. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. Current libraries are a part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We were also interested in the activity of promoters during toxin overexpression. We used a targeted RNA-sequencing approach that allowed us to map the distinct 5- and 3-ends of toxin-cleaved RNA and also the beginnings of transcripts. We extracted total RNA from cultures where the expression of MazF or MqsR was induced; RNA from log phase culture was used as the control. In addition, RNA extracted from stationary phase culture was used to test for the possible toxin cleavage in natural stress conditions. The cleaved RNA is rapidly recycled making it possible that we miss important cleavage sites due to RNA degradation during the prolonged stationary phase. Therefore, in addition to wt E. coli we also used stationary phase RNA from exoribonuclease deficient strain where RNA cleavage products accumulate. Identification of the 5 ends is based on ligation of RNA adapters to the cellular RNA molecules, which requires 5 -monophosphates. Mapping of the 3 -ends is based on poly(A) tailing of RNA, which requires 3 -OH groups. Transcription initiation, ordinary cellular RNases, and toxin endonucleases all produce different types of RNA ends. These can be enzymatically converted to 5 -phosphates and 3 -OHs, which allows one to separately quantify the RNA ends produced by each of these processes from a single biological sample. Briefly, (i) primary transcripts have 5 -triphosphate ends that can be converted to monophosphate by Tobacco Acid Pyrophosphatase (TAP), and 3 -hydroxyl ends that can be directly polyadenylated by poly(A) polymerase. (ii) Most cellular RNases (excluding RNase I and the toxins) produce 5 -monophosphates and 3 -hydroxyls, which are directly usable for ligation/polyadenylation. (iii) RNase I, MazF, and several other toxins produce 5 -hydroxyl ends that need to be phosphorylated by T4 Polynucleotide Kinase (PNK) prior to ligation, and 2,3 -cyclic phosphates that need to be dephosphorylated and converted to 3 -hydroxyls, also by T4 PNK. In short, for each of our RNA samples three 5 end and two 3 end libraries were made. The PNK treated libraries contain reads mapping specifically to 5- or 3-ends left by toxin (or RNaseI) cleavage and TAP treated libraries have extra reads mapping to beginnings of the transcripts.
Project description:Bacteria possess many small noncoding RNAs whose regulatory roles in pathogenesis are little understood due to a paucity of macroscopic phenotypes in standard virulence assays. Here, we use a novel Dual RNA-seq approach for a single-step simultaneous RNA profiling in both pathogen and host to reveal molecular phenotypes of sRNAs during infection with Salmonella Typhimurium. We identify a new PhoP/Q-activated small RNA which upon bacterial internalization acts to temporally control the expression of both, invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity is shown to adjust the human response to replicating Salmonella, and have a pervasive impact on host RNA expression both inside and outside protein-coding regions including infection-specific alterations of an array of long noncoding RNAs. Our study provides a paradigm for a comprehensive RNA-based analysis of intracellular bacterial pathogens without their physical purification from a host and a new discovery route for hidden functions of pathogen genes. Comparative RNA-seq (in vitro medium shift experiment)
Project description:Mycobacterium tuberculosis is an intracellular human pathogen with the ability to resist and adapt to many adverse conditions it encounters upon infection. Among these, overcoming the production of nitric oxide by macrophages could be key for M. tuberculosis success. We have challenged M. tuberculosis with a sub-lethal concentration of nitric oxide and followed the transcriptomic response through RNA-seq for 48 hours.
Project description:Bacterial transcription is controlled by many transcription factors and promoters. Currently many promoters are mapped only in in vitro conditions. We use an intra-macrophage model to map the promoters and their expression in vivo. Transcriptome analysis of S.Typhimurium 4/74 within macrophages using RNA-seq in biological replicates. dRNA-seq also performed in biological replicates.
Project description:Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26-nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the argonaute ERGO-1, DICER, and a series of associated (ERI) factors. This primary process leads to production of a much more abundant class of 22-nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely-structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA. 24 small RNA and 2 polyA RNA samples
Project description:Cellular RdRPs play a critical role in the development of many organisms. Using high-throughput small RNA and messenger RNA sequencing we found that the Caenorhabditis elegans RdRP, EGO-1, is required to produce small RNAs antisense to a number of germline-expressed genes through several developmental stages. We found that these genes fall into distinct classes including genes required for kinetochore and nuclear pore assembly, as well as the production of histone-modifying and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong correlation between the loss of small RNAs and the rise of mRNA levels in ego-1 mutant animals. 5'-monophosphate-independent small RNA sequencing from ego-1(om84) and control L3, L4, and adult C. elegans hermaphrodites
Project description:New tools for studying bacterial transcripts at the single nucleotide level offer an unparalleled opportunity to understand the bacterial transcriptome. For the model pathogen Salmonella enterica serovar Typhimurium, it is necessary to identify the regulatory inputs for all RNA transcripts, including small RNAs (sRNAs) and coding genes. Here, we use RNA-seq to define the transcriptomes of mutants lacking 18 global regulatory systems that, among other functions, modulate the expression of the SPI1 and SPI2 Type Three secretion systems in S. Typhimurium strain 4/74. We directly compared the roles of the major regulators of transcription, and reported the effects of the regulatory mutations on expression of sRNAs. We also use this method to describe the impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts. Transcriptome analysis of S. Typhimurium 4/74 using RNA isolated wild-type and mutants grown under infection-relevant conditions.
Project description:Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium TMC724. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these coincided with the start codons and therefore belong to leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 9 intergenic small RNAs were mapped. Four of the revealed intergenic small RNAs, including igMAV_1034-1035 expressed at a very high level, have no homologs in M. tuberculosis, whilst M. avium lacks several intergenic sRNAs present in M. tuberculosis. Among those, MTS479 and MTS1338 are of special interest due to their possible implication in pathogenesis. Elucidation of differences in the repertoire of intergenic sRNAs between the two mycobacterial species may improve our understanding of mycobacterial diseases pathogenesis. Transcriptional profile of Mycobacterium avium TMC724, grown at 37°C in Dubos broth until mid-logarithmic growth phase
Project description:Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing though formation of RNA duplexes. While progress has been made in identifying the capabilities of siRNAs in silencing of foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a C. elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development, and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. For a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role for the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis Sequencing of small RNAs from a population of C. elegans isolated spermatogenic cells and from two paired sets of rrf-3 mutant and control whole males