Download Sinorhizobium meliloti YbeY: a novel endoribonuclease involved in

Sinorhizobium meliloti YbeY: a novel endoribonuclease involved in RNA-mediated
gene silencing
José I. Jiménez-Zurdo, Marta Robledo, Alexandra Peregrina, Natalia I. García-Tomsig, Margarida
Saramago1, Rute G. Matos1, Cecilia M. Arraiano1, Rolf Hilker2, Anke Becker2
Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada,
Spain
1Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras,
Portugal
2LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps Universität, Marburg,
Germany
[email protected]
The ybeY gene integrates the proposed minimal prokaryotic genome. Structural studies of YbeY
orthologs revealed a conserved three histidine H(X)3H(X)4DH motif shared by metallo-hydrolases and
global similarity to the MID domain of eukaryotic AGO proteins involved in RNA-directed gene
silencing. These features suggest that YbeY could serve catalytic and/or Hfq-like RNAbinding/chaperone functions in bacteria, as suggested by previous studies in E. coli and the legume
symbiont Sinorhizobium meliloti (1-3). We have biochemically and genetically characterized the YbeY
ortholog of S. meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY
yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a
similar experimental setup (4). Purified SmYbeY behaved as a monomer that indistinctly cleaved
double- and single-stranded RNA substrates, a unique ability among bacterial endoribonucleases.
SmYbeY-mediated catalysis was supported by the divalent metal ions Mg 2+, Mn2+ and Ca2+, which
influenced differentially on cleavage efficiency and reactivity patterns. Ca 2+ specifically impaired
SmYbeY activity on structured RNA molecules. SmYbeY loss-of-function compromised expression of
housekeeping genes related to core energy and RNA metabolism, whilst promoting accumulation of
motility, transport and late symbiotic transcripts. Some of the upregulated mRNAs might be SmYbeY
substrates which are targeted by sRNAs that bind Hfq. A genetic reporter assay confirmed that
SmYbeY participates in sRNA-mediated down-regulation of the amino acid ABC transporter prbA
mRNA. We have thus discovered a bacterial endoribonuclease with unprecedented catalytic features,
acting also as novel silencing enzyme in riboregulation.
(1) Gil et al. (2004) Mol. Biol. Rev. 68:518-537.
(2) Pandey et al. (2011) Nucleic Acids Res. 39:4691-4708.
(3) Jacob et al. (2013) Mol. Cell 49:427-438.
(4) Torres-Quesada et al. (2014) RNA Biol. 11:563-579.
This work was funded by ERDF-cofinanced grants AGL2009-07925, CSD2009-00006 and BFU201348282-C2-2-P from MINECO.