Sinorhizobium meliloti RNase III: Catalytic Features and Impact on Symbiosis.

Margarida Saramago,José I Jiménez-Zurdo,Cecília M Arraiano,Rute G Matos,Marta Robledo

doi:10.3389/fgene.2018.00350

Abstract

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its α–proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg2+ as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants.

Highlights

Ribonucleases (RNases) promote processing and degradation of RNA transcripts to rapidly adjust gene expression according to cellular needs
The SmRNase III mutant variants E125A and E125Q were largely inactive on R1.1 in the presence of Mg2+, but retained partially cleavage ability at site a of the substrate when Mn2+ was used as cofactor (Figure 2A; right panels). These results indicate that R1.1 cleavage in reactions involving the wild-type enzyme was protein-specific, further suggesting a major role of the ultraconserved E125 amino acid in the metal-dependent catalysis mediated by SmRNase III
Our data showed that SmRNase III demands significantly higher concentrations of both Mg2+ (10 mM) and Mn2+ (1–50 mM) for R1.1 depletion

Summary

Introduction

Ribonucleases (RNases) promote processing and degradation of RNA transcripts to rapidly adjust gene expression according to cellular needs. RNase E is regarded as the major single-strand specific endoribonuclease in gram-negative bacteria (Silva et al, 2011). In S. meliloti, this RNase was shown to be required for small RNA (sRNA)-mediated post-transcriptional silencing of quorumsensing and cell cycle related mRNAs (Baumgardt et al, 2014, 2016; Robledo et al, 2015). In gram-positive bacteria, RNA degradation initiates with RNase Y and RNase J1/2 endoribonucleases (Laalami et al, 2014; Durand et al, 2015). The ultraconserved E. coli YbeY protein was characterized as a novel single-strand specific endoribonuclease, whose activity affects ribosome quality control and processing of 16S rRNA 3 -terminus (Jacob et al, 2013). S. meliloti YbeY was shown to promote degradation of mRNAs from transporter genes upon their antisense interaction with the AbcR2 sRNA (Saramago et al, 2017)

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