Mechanism of 3' Exonucleolytic mRNA Turnover in Bacillus Subtilis

<p>PROJECT SUMMARY: Our laboratory studies the essential process of mRNA decay in the model Gram-positive bacterium, Bacillus subtilis. Rapid turnover of mRNA fragments is required to replenish ribonucleotidepools and to avoid non-productive translation on mRNA fragments. Relative to what is known about bacterialtranscription and translation, much less is known about mRNA decay. All bacterial species contain a 3'-to-5'exoribonuclease, which degrades mRNA fragments that are generated by a decay-initiating endonucleasecleavage. Much evidence suggests that the key 3'-to-5' exonuclease in B. subtilis is polynucleotidephosphorylase (PNPase), encoded by the pnpA gene. RNA-Seq data show that 5'-proximal RNA fragmentsfor hundreds of genes accumulate in a pnpA deletion strain. The current proposal aims to understand thespecificity of mRNA turnover by PNPase and what compensates for PNPase in a pnpA deletion strain. Structural studies suggest that RNA is threaded from its 3' end into a central channel of PNPase, which canonly bind single-stranded RNA. Thus, efficient decay of mRNA that contains secondary structure may dependon RNA helicase activity. Preliminary data show that the major B. subtilis RNA helicase, CshA (encoded bythe cshA gene) is required for rapid decay of some mRNA fragments. We propose to use new RNA-Seqprotocols in B. subtilis ∆pnpA and ∆cshA strains to discover the nature of mRNA sequences that determinesusceptibility to PNPase and dependence on CshA. The results of RNA-Seq experiments will guide geneticand biochemical experiments that probe the requirements for efficient PNPase-mediated decay. Availabletools will be used to explore the relationship of PNPase-mediated decay to RNase Y (the major decay-initiatingendonuclease), to ribosome flow, and to the ribosome rescue system. In addition, a unique screening methodfor determining susceptibility to decay is proposed. The screen promises to give insight into the nature of RNAsequences that are efficiently degraded by PNPase, and when there is a need for helicase activity. Despite considerable accumulation of RNA fragments, a pnpA knockout strain grows well, indicating thatsome 3' exonuclease compensates for the loss of PNPase. The other three known 3' exoribonucleases of B.subtilis are likely not significantly involved in mRNA turnover. In preliminary experiments, we detect at leastone 3' exonuclease activity in an extract of a strain that is missing all four of the known 3' exonucleases. Theidentity of this activity will be pursued using a biochemical approach, and its function in mRNA turnover will bestudied. Discovery of an additional processive 3' exonuclease, which is not predicted from genome sequence,will impact greatly on our understanding of bacterial RNA processing and decay.RELEVANCE: Degradation of messenger RNA is an essential function of bacteria. A thorough understandingof the mechanism of mRNA decay will enable design of antimicrobial agents that disrupt this process andthereby interfere with bacterial cell growth.</p>