Analysis of the cross-talk between transcriptional and post-transcriptional small RNA pathways in Arabidopsis – RNACROSSTALK

A-Résumé du projet - - Small RNAs, 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in a number of processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage, and translational repression. To date, four distinct classes of endogenous small RNAs have been identified in plants: i) microRNAs (miRNAs), ii) trans-acting siRNAs (ta-siRNAs), iii) natural cis-antisense RNA (nat-siRNAs), and iv) heterochromatin siRNAs (hc-siRNAs). miRNAs, ta-siRNAs and nat-siRNAs repress gene expression at the post-transcriptional level (posttranscriptional gene silencing-PTGS), whereas hc-siRNAs control transposable elements at the transcriptional level (transcriptional gene silencing-TGS) and participate in heterochromatin assembly and maintenance. - In the plant model Arabidopsis, the four classes of small RNAs are produced by four distinct DICER-LIKE RNaseIII enzymes (DCL). Arabidopsis also contains five double-stranded RNA binding proteins (DRB), which assist DCLs in the processing of dsRNAs, and ten ARGONAUTE proteins (AGO), which associate with small RNAs and cleave complementary RNAs (PTGS) or guide chromatin remodeling (TGS). DCL1, DRB1 and AGO1 act in the miRNA pathway, whereas DCL4, DRB4, AGO1 and AGO7 act in the ta-siRNA pathway. The nat-siRNA pathway involves DCL2, and the hc-siRNA pathway involves DCL3 and AGO4. The function of the other DRBs and AGOs is unknown. - A cross-talk between TGS and PTGS has been established in worms where the regulation of RNAi (the animal counterpart of PTGS) involves several chromatin genes. In plants, two chromatin genes have been implicated in the control of transgene-induced PTGS (work of Partner1). In addition, some miRNAs, as well as PTGS-related siRNAs, direct DNA methylation of their targets. However, the regulation and the biological role of this process are not understood. Recently, Partner1 has shown that the PTGS/ta-siRNA pathway component SGS3 interacts with chromatin proteins, reinforcing the idea of a cross-talk between TGS and PTGS. Partner2 has shown that RNAs derived from SINE retroposons, which are controlled by the hc-siRNA pathway, interact with DRB1, which participates in the miRNA pathway. Partner2 also has shown that ectopic expression of SINE elements leads to developmental defects that can be rescued by over-expressing DRB4, which participates in the ta-siRNA pathway, indicating additional connections between TGS and PTGS pathways. - In this project, we will investigate the cross-talk between TGS and PTGS pathways in plants through a combination of genetic and biochemical approaches: - - Regulations of MIR, TAS and SINE genes by the TGS machinery will be explored by examining the accumulation of mature miRNAs, ta-siRNAs, PTG-Silenced transgene-siRNAs, and SINE hc-siRNAs, as well as their precursors, in a series of TGS mutants, including all known TGS mutants and mutants impaired in their paralogs, mutants of SGS3 chromatin partners, and double and triple derivative mutants. This approach will extend previous work on transgene-siRNAs to endogenous small RNAs, will uncover the role of SGS3 and chromatin interactions, and will decipher the contribution of Pol II, Pol III and Pol IV transcription in SINE hc-siRNA production. - - The regulation of SINE elements by the PTGS machinery will be determined by analyzing the accumulation of SINE intermediates and mature hc-siRNAs in all known PTGS mutants as well as mutants impaired in their paralogs and in double and triple derivative mutants. This approach will expand previous work on the maturation of SINE RNAs and will provide additional functions for AGO and DRB proteins. We will express TAP-tagged versions of the five DRBs, purify RNP complexes via immunoprecipitation, and characterize their protein and RNA compositions. This biochemical analysis will implicate DCL/DRB complexes i...