Genome-Wide Discovery of Small RNAs in Mycobacterium tuberculosis

Abstract

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5′/3′ UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5′ end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

“…Many of the published putative sRNAs failed to reach our depth and boundary criteria designed to distinguish them from mRNA degradation products, but differences also likely reflect alterations in culture conditions, growth phases, and methods of library preparation. However, where the 5′ and 3′ boundaries of sRNAs have been experimentally determined by 5′ and 3′ rapid amplification of cDNA ends (RACE), there was excellent concordance with our analysis (45, 48–50). Of the 14 experimentally mapped M. tuberculosis sRNA ends, BS_finder mapped 12 within 3 bp of the experimentally defined end (Fig.…”

Revealing of Mycobacterium marinum Transcriptome by RNA-seq

“…As a result, 74 and 38 intergenic transcripts were found out in log phase culture and early stationary phase culture separately (Table S9). All these transcripts were blasted against known sRNAs reported in [33,34], and no significant matches were found (E<10 -5 ). This is probably attributed to the reason that we manually enriched cDNA with size mainly between 100 and 550bp before sequencing, so those transcripts with small size (<100nt) could not be detected in our results.…”

RNA-Seq Analysis of Mycobacterium avium Non-Coding Transcriptome

“…For instance, Pellin et al performed high-throughput sequencing of sRNA fractions of M. tuberculosis and identified ∼2,000 sRNA candidates by combining information regarding the read coverage with conservation analysis of intergenic regions [18]. A subsequent microarray analysis further confirmed the expression of 258 of these sRNA candidates, including 22 intergenic sRNAs and 152 antisense RNAs [19].…”