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| Status |
Public on Jan 15, 2025 |
| Title |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [fiveprime_end_seq] |
| Organism |
Bacillus subtilis |
| Experiment type |
Other
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| Summary |
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.
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| Overall design |
RNA 5′/3′-end sequencing and Rend-seq were performed on B. subtilis strains deficient in exonucleolytic activity. These datasets, combined with similar measurements performed on strains with an additional knockouts of individual endoribonucleases, were used to determine the locations of endoribonucleolytic cleavage transcriptome-wide.
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| Contributor(s) |
James T, Julia D, Hannah L, Jean-Benoît L, Sylvain D, Frédérique B, Ciarán C, Gene-Wei L |
| Citation(s) |
39883015 |
| |
| Submission date |
Aug 16, 2024 |
| Last update date |
Feb 20, 2025 |
| Contact name |
James Christopher Taggart |
| E-mail(s) |
james_taggart@hms.harvard.edu
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| Organization name |
Harvard Medical School
|
| Department |
Systems Biology
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| Lab |
Allon Klein
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| Street address |
200 Longwood Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
| GPL24109 |
Illumina NextSeq 500 (Bacillus subtilis) |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE275083 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis |
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| Relations |
| BioProject |
PRJNA1149056 |
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