Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus

Abstract

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y. Author summaryIn order to correctly regulate the level of RNAs, bacteria require their RNA to be continuously synthesised and degraded. However, even related bacterial species can have different sets of ribonucleases, each with their own target criteria. Here we explore which sequence elements of an RNA are important for being targeted by the endoribonuclese RNase Y in the two bacteria, Staphylococcus aureus and Bacillus subtilis. We specifically examine the RNase Y dependent cleavage of two transcripts that have homologs in both bacteria. We identify a short single-stranded regions immediately downstream of the cleavage position can be modified to change the cleavage efficiency up to 20-fold. We furthermore discover that a secondary structure a few nucleotides downstream of the cleavage is required for cleavage and that the positioning of the cleavage can be modulated by moving this structure.