Abstract
RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference.
Highlights
Ribonucleic acid (RNA) silencing is triggered by doublestranded RNA, processed into 21- to 24-nt small interferingRNA or microRNA by RNaseIII-like enzymes called Dicers, or Dicer-like (DCL) in plants [1,2,3]
To identify new factors required for RNAi, we established an inverted repeat (IR) construct corresponding to the 5 region (‘LU’) of the firefly LUCIFERASE (LUC) gene driven by the phloem specific AtSUC2 promoter
35S promoter-driven expression of the Tomato bushy stunt virus P19 silencing suppressor led to a strong luminescence recovery in the p35S:LUC construct (PL), supporting post-transcriptional silencing mediated by LUderived 21-nt siRNAs, for which P19 displays high and selective affinity (Supplementary Figure S1J–L) [29]
Summary
Ribonucleic acid (RNA) silencing is triggered by doublestranded RNA (dsRNA), processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers, or Dicer-like (DCL) in plants [1,2,3]. Small RNAs guide ARGONAUTE (AGO)-containing RNA-induced silencing complexes (RISCs) to suppress target gene expression at the level of transcription, RNA stability or translation. In Arabidopsis, 21-nt siRNA and miRNA mainly incorporate into AGO1 to promote cleavage or translational inhibition of target transcripts [4,5], whereas 24-nt siRNAs guide heterochromatin formation by recruiting AGO4, AGO6 or AGO9 [6]. The effect of RNAi can extend beyond the sites of its initiation, owing to the movement of signal molecules with defensive and developmental roles [7,8]. The spread of virusderived siRNA (vsiRNA) most likely immunizes surrounding cells that are yet to be infected [12], whereas movement of endogenous trans-acting (ta)siRNA generates a gradient of target gene expression participating in organ polarization [13,14]
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