Petit ARN ciblant la séquence codante de l’ARNm : régulation négative d’expression par déstabilisation de l’ARNm en absence de répression de la traduction – Target: CdS
sRNA targeted to the mRNA coding sequence: mRNA destabilization in the absence of translational repression
The molecular mechanism underlying the regulatory pathway(s) mediated by trans-encoded sRNA in Escherichia coli
The molecular mechanism underlying the regulatory pathway(s) mediated by trans-encoded sRNA in Escherichia coli
The potential of RNA molecules to exert regulatory functions in conjunction with auxiliary proteins has only recently been recognized and has emerged as a major field in molecular biology and biomedical research. In enterobacteria, such molecules constitute an ever-growing class of regulators of stress responses, metabolism and virulence. The majority of the known bacterial trans-encoded small RNAs (sRNAs) negatively regulate their target mRNAs by base pairing. This interaction leads to inhibition of translation and/or degradation of mRNA reducing the protein levels of the target gene. Whilst the first mechanism has ample experimental support, the messenger destabilization process, which is often regarded as a downstream consequence of generating untranslatable messengers, remains controversial in the absence of supporting data. However, a recent study strongly questions this assumption by reporting Salmonella MicC-ompD as the first example of RNase E dependent destabilization of an mRNA without primary effects on translation.<br />After a decade of intensive research, we are beginning to have a full appreciation of the importance and prevalence of sRNAs in numerous and diverse means of gene regulation. Nevertheless, the molecular mechanism underlying sRNA-induced mRNA decay is largely unknown and has yet to be characterized in significant detail. The proposed research will add to our knowledge to explore it by using in vivo and in vitro experimental approaches. <br />
The particular case of RybB-induced regulation of ompC mRNA was our model. The objectives of the project were first to determine how RybB promotes the degradation of the ompC mRNA by targeting its 5’ end hairpin.
The bacterial trans-encoded small RNAs (sRNAs) achieve their tasks by base pairing their target mRNAs with imperfect complementarities. sRNAs have repeatedly been shown to repress bacterial mRNAs translation by masking the ribosome binding site (RBS). This inhibits mRNA interactions with 30S ribosomes primarily by competition with 16S rRNA binding, and consequently, translation initiation. The RNA-RNA duplex formation is often associated with the decay of both RNA species. In enterobacteria, the primary nuclease through which sRNAs trigger transcript instability is RNase E. This destabilization that is often regarded as a downstream consequence of generating untranslatable messengers has been proposed to be the primary regulatory mechanism, as many of the recently identified regulatory sRNAs have no obvious anti-RBS region, indicating that other complementary mRNAs might be repressed without the canonical block of ribosome binding. A first example of sRNA-guided mRNA inactivation without primary effects on translation was reported for the Salmonella MicD-ompD couple with a targeting of the mRNA coding sequence. Here, we present evidence for the RybB-ompC couple in Salmonella and E. coli suggesting that the pairing of RybB into a 5’ hairpin of ompC mRNA entails RNase E-dependent mRNA processing and destabilization, and can down-regulate this target without a concomitant block of translation. The adequacy of our results with the recently published work providing evidence of how sRNA catalyzed selective degradation of target mRNA will be discussed.
Future analysis must determine how and where RNase E acts on ompC and presumably other targets following RybB pairing. At current, both the release of the 5’ hairpin and the observed mRNA processing downstream of the RybB-ompC duplex are in favor of a model in which RybB inactivates ompC mRNA by removing its 5’ stabilizer. Given that processing maps to a single-stranded A-rich region, RNase E is not unlikely to cleave directly at the -38 position of ompC. However, the determined stable 3’ end may also result from inhibition of 3’-5’ exonucleolytic activity by the RybB-ompC duplex, similar to observations with REP elements in E. coli 46. We are currently trying to establish of an in vitro system to determine the individual steps of RybB-induced target decay.
-1 research article is in preparation for publication in a peer-reviewed journal:
Bouvier, M., Sharma, C.M., Carpousis, A.J. and Vogel, J. RybB promotes ompC mRNA destabilization in an RNase E-dependent fashion by targeting of a stability element.
-1 review and 1 MicroCommentary on subjects related to the ANR project were also published:
Bouvier, M., and Carpousis, A.J. (2011). A tale of two mRNA degradation pathways mediated by RNase E. Mol Microbiol 82, 1305–1310.
Bandyra, K.J., Bouvier, M., Carpousis, A.J., and Luisi, B.F. (2013). The social fabric of the RNA degradosome. Biochim Biophys Acta 1829, 514–522.
La capacité des molécules d’ARN à exercer des fonctions régulatrices en partenariat avec des protéines auxiliaires a été découverte récemment et a émergé comme un domaine majeur de la biologie moléculaire et de la recherche biomédicale. Chez les entérobactéries, ces molécules constituent une classe grandissante de régulateurs impliqués dans la réponse au stress, le métabolisme et la virulence. La majorité des petits ARNs bactériens régule négativement l’expression de leur cible, les ARN messagers (ARNm), en s’appariant avec eux. Cette interaction entraîne une inhibition de la traduction et/ou une dégradation de l’ARNm ayant pour conséquence une répression de sa synthèse protéique. Alors que le premier mécanisme est amplement supporté expérimentalement, la déstabilisation du messager, souvent considéré comme secondaire par la création d’un ARNm non traductible, est un processus mal caractérisé. Ce dernier point peu étayé par des données tangibles reste controversé. Toutefois, une étude récente sur MicC-ompD chez Salmonella remet sérieusement en question cette hypothèse en rapportant le premier le cas de déstabilisation d’un ARNm sans effets primaires sur la traduction.
Après une décennie de recherche intensive, nous commençons à avoir une appréciation complète de l'importance et de la prévalence des petits ARN régulateurs dans de nombreuses voies de régulation de l’expression des gènes. Néanmoins, le mécanisme moléculaire sous-jacent est largement incompris et reste à caractériser en détail. Ce projet propose d’améliorer notre connaissance de ce mécanisme en utilisant des approches à la fois in vivo et in vitro.
Coordination du projet
Marie BOUVIER (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE MIDI-PYRENEES)
L'auteur de ce résumé est le coordinateur du projet, qui est responsable du contenu de ce résumé. L'ANR décline par conséquent toute responsabilité quant à son contenu.
Partenariat
LMGM CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE MIDI-PYRENEES
Aide de l'ANR 120 587 euros
Début et durée du projet scientifique :
- 36 Mois
