A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [mpra_quant]
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. Overall design: A library of plasmids encoding barcoded RNAs with mutated variants of RNase Y cleavage sites was constructed. This library was introduced to B. subtilis and both RNA and genomic DNA was harvested. To determine the efficiency of cleavage at each cleavage site variant, barcodes were PCR amplified and quantified by sequencing. A separate amplicon was sequenced to map barcodes to variant sequences.