# Mechanism of 3' exonucleolytic mRNA turnover in Bacillus subtilis

> **NIH NIH R01** · ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI · 2021 · $339,000

## Abstract

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 ribonucleotide
pools and to avoid non-productive translation on mRNA fragments. Relative to what is known about bacterial
transcription 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 endonuclease
cleavage. Much evidence suggests that the key 3'-to-5' exonuclease in B. subtilis is polynucleotide
phosphorylase (PNPase), encoded by the pnpA gene. RNA-Seq data show that 5'-proximal RNA fragments
for hundreds of genes accumulate in a pnpA deletion strain. The current proposal aims to understand the
specificity 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 can
only bind single-stranded RNA. Thus, efficient decay of mRNA that contains secondary structure may depend
on RNA helicase activity. Preliminary data show that the major B. subtilis RNA helicase, CshA (encoded by
the cshA gene) is required for rapid decay of some mRNA fragments. We propose to use new RNA-Seq
protocols in B. subtilis ∆pnpA and ∆cshA strains to discover the nature of mRNA sequences that determine
susceptibility to PNPase and dependence on CshA. The results of RNA-Seq experiments will guide genetic
and biochemical experiments that probe the requirements for efficient PNPase-mediated decay. Available
tools will be used to explore the relationship of PNPase-mediated decay to RNase Y (the major decay-initiating
endonuclease), to ribosome flow, and to the ribosome rescue system. In addition, a unique screening method
for determining susceptibility to decay is proposed. The screen promises to give insight into the nature of RNA
sequences 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 that
some 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 least
one 3' exonuclease activity in an extract of a strain that is missing all four of the known 3' exonucleases. The
identity of this activity will be pursued using a biochemical approach, and its function in mRNA turnover will be
studied. 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 understanding
of the mechanism of mRNA decay will enable design of antimicrobial agents that ...

## Key facts

- **NIH application ID:** 10074571
- **Project number:** 5R01GM125655-04
- **Recipient organization:** ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
- **Principal Investigator:** DAVID H BECHHOFER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $339,000
- **Award type:** 5
- **Project period:** 2017-12-15 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10074571

## Citation

> US National Institutes of Health, RePORTER application 10074571, Mechanism of 3' exonucleolytic mRNA turnover in Bacillus subtilis (5R01GM125655-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10074571. Licensed CC0.

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