Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation.
Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium 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. Purified SmYbeY behaved as a monomer that indistinctly cleaved single- and double-stranded RNA substrates, a unique ability among bacterial endoribonucleases. SmYbeY-mediated catalysis was supported by the divalent metal ions Mg2+, Mn2+ and Ca2+, which influenced in a different manner cleavage efficiency and reactivity patterns, with Ca2+ specifically blocking activity on double-stranded and some structured RNA molecules. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Some of the latter transcripts are known Hfq-binding sRNA targets and might be SmYbeY substrates. Genetic reporter and in vitro assays confirmed that SmYbeY is required for 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 gene silencing enzyme.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Oeiras, Portugal.
Margarida
M
Saramago
Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
Alexandra
A
Peregrina
Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
Marta
M
Robledo
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Oeiras, Portugal.
Rute G
RG
Matos
LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-University Marburg, Marburg, Germany.
Rolf
R
Hilker
LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-University Marburg, Marburg, Germany.
Javier
J
Serrania
LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-University Marburg, Marburg, Germany.
Anke
A
Becker
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Oeiras, Portugal.
Cecilia M
CM
Arraiano
Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
José I
JI
Jiménez-Zurdo
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002876:Chromosomes, Bacterial / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D020868:Gene Silencing; D005798:Genes, Bacterial; D017930:Genes, Reporter; D035001:Host Factor 1 Protein / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013559:Symbiosis / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D035001:Host Factor 1 Protein; D008667:Metalloproteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D004722:Endoribonucleases