Unknown,Transcriptomics,Genomics,Proteomics

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Transcript 5'-end mapping was used to identify transcriptional start sites and RNA processing sites genome-wide in Mycobacterium tuberculosis

ABSTRACT: Most eukaryotic mRNAs are enzymatically processed before they are translated, but less is known about mRNA processing in prokaryotes. While bacterial rRNAs and tRNAs are known to be highly processed, only a handful of bacterial mRNAs have been shown to be processed by ribonucleases, typically altering transcript stability. We hypothesized that mRNA processing might be more widespread in prokaryotes and constitute a previously underappreciated layer of post-transcriptional regulation. We therefore adapted RNA-seq methodologies to map transcript 5’ ends across the Mycobacterium tuberculosis transcriptome, distinguishing between transcriptional start sites (TSSs) and RNA processing sites. We report for the first time genome-wide mRNA endonucleolytic cleavage patterns in a prokaryote, and find that mRNA processing is widespread in M. tuberculosis. This has physiologic consequences, as mRNA cleavage plays a regulatory function in the ESX-1 locus by producing a transcript fragment encoding the secreted proteins EsxB and EsxA that is significantly more stable than the parent transcript, leading to greater steady-state transcript abundance. At least 20 other loci display similar patterns of processing-associated differences in transcript levels in M. tuberculosis. Mycobacteria therefore use mRNA processing to effect differential abundance of sub-sections of polycistronic transcripts, which may provide additional layers of regulation. Moreover, our results demonstrate that stable, processed mRNAs are a ubiquitous feature in this prokaryotic transcriptome, suggesting that mRNA processing may represent a major post-transcriptional regulatory mechanism. RNA from two biological replicate cultures was analyzed by RNA-seq for expression analysis and 5'-end-mapping for identification of transcriptional start sites and RNA processing sites. An additional set of three biological replicate cultures were analyzed by RNA-seq for expression analysis using a different library construction method.

ORGANISM(S): Mycobacterium tuberculosis

SUBMITTER: Scarlet Shell

PROVIDER: E-GEOD-62152 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells. Series includes three datasets of total small RNA reads from wild type and Dicer negative 293T cells. Also included are total small RNA reads of the Dicer-negative cell line NoDice(4-25) transfected with a vector expressing human Dicer, Drosophila Dicer1, or mock transfected. RISC-associated small RNAs identified by ribonucleoprotein immunoprecipitation (RIP-Seq) in wild type 293T, Dicer-negative NoDice(4-25) line, and NoDice(4-25) transfected with either hsa-miR-155 or kshv-miR-K12-11 miRNA duplexes. The final data series are PAR-CLIP libraries which identified microRNA targets in untransfected NoDice(4-25) cells and NoDice(4-25) cells transfected with either hsa-miR-92a, hsa-miR-155, kshv-miR-K12-11 miRNA duplexes

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Transcript 5'-end mapping was used to identify transcriptional start sites and RNA processing sites genome-wide in Mycobacterium tuberculosis

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