Redox regulation of the RNAse III like protein RTL1 and its impact on small RNAs biogenesis.
Résumé
Plants are sessile organisms confronted to biotic or abiotic constraints. One of the main cellular response to stresses is modification of the redox homeostasis, which can induce signaling response pathways, for example by altering the redox status of thiol residues in proteins. In eukaryotic cells, small RNAs (siRNAs and miRNAs) are major regulators of gene expression, involved in most developmental and stress-response processes. Their biogenesis is mediated by RNaseIII endonuclease enzymes called DICER-LIKE (DCL) and RNASE THREE-LIKE (RTL), which mature almost all classes of double-stranded RNA precursors. In Arabidopsis thaliana, it has been shown that the RNA cleavage activity of the RTL1 isoform depends on the oxidation state of a specific cysteine (C230) located in its RNA binding domain (RBD). To better understand the role of cysteines on RTL1 activity, we have constructed Cys substitution versions of RTL1 preventing or mimicking oxidation. These versions have been tested biochemically for their RNA binding activities. MicroScale thermophoresis (MST) assays show that the Kd of RTL1 with mutated catalytic site (RTL1-cat) is in the nM range (260-670). In contrast, deletion of the RBD or mutation of the C230 (in S230), increases the Kd of RTL1-cat to the µM range (2-5). Thus, MST assays suggest that C230 may contribute to the stabilization of RTL1-RNA binding interactions and then facilitate RNA cleavage activity. In parallel, we have evaluated the contribution of RTL1 cys-oxidation to IR-PTGS triggered by overexpressing Cys substitution versions of RTL1 in the context of the JAP3 construct expressing PDS dsRNA under the control of the phloem-specific SUC2 promoter.