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A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [rend_seq]

ABSTRACT: RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches – transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites – that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5′- and 3′-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features, each with mild contributions. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE275076 | GEO | 2025/01/15

REPOSITORIES: GEO

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A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [fiveprime_end_seq]

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Bacterial RNase III cleaves long dsRNA at preferred sites

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2011-08-31 | E-GEOD-30430 | biostudies-arrayexpress

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Project description:The narrow-specificity endoribonuclease RNase III and the 5’ exonuclease RNase J1 have been recently found to be not essential in the Gram-positive model organism, Bacillus subtilis. In this study, we performed a global analysis of internal 5’ ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5’ monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions were direct targets of RNase III. A set of regular RNA-seq experiments were performed to investigate RNA profiles in these strains and used to account for the changes in RNA abundance indirectly caused by the loss of RNase III in PARE. The PARE analysis also revealed an accumulation of 3’-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. In addition, an endonuclease cleavage just two nucleotides downstream of the 16S rRNA 3’ end was discovered with PARE analysis. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic vs. endonucleolytic nature of 16S rRNA maturation

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