Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

El Mouali, Youssef, Ponath, Falk, Scharrer, Vinzent, Wenner, Nicolas, Hinton, Jay CD and Vogel, Joerg (2021) Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease. RNA, 27 (12). pp. 1512-1527.

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Abstract

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of <i>Salmonella</i> with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

Item Type:	Article
Uncontrolled Keywords:	RNA-binding proteins, ProQ, Hfq, Lon protease, protein stability
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User:	Symplectic Admin
Date Deposited:	31 Jan 2022 16:46
Last Modified:	18 Jan 2023 21:14
DOI:	10.1261/rna.078954.121
Open Access URL:	https://rnajournal.cshlp.org/content/27/12/1512.lo...
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3147890