HHMI Investigator and Carlos O. MIller Professor Howard Hughes Medical Institute, Indiana University
In plants, RNA-dependent DNA methylation (RdDM) leads to transcriptional silencing of transposons, DNA viruses and a subset of genes. The process begins with transcription of DNA into double-stranded (ds) RNA by the coupled activities of nuclear multisubunit RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). DICER-LIKE3 (DCL3) then cuts the dsRNAs into short interfering RNAs that are diverse in sequence and can be 23 or 24nt in length. Using purified Pol IV, RDR2 and DCL3, we have recapitulated siRNA biogenesis in the testtube, revealing the intriguing and surprising reaction mechanisms of the enzymes involved. Our results show that Pol IV and RDR2 activities are mechanistically coupled and impart on their transcripts molecular attributes that specify how they are processed by DCL3, and subsequently loaded into ARGONAUTE 4 to generate RNA-guided silencing complexes. Collectively, these reaction mechanisms account for the channeling of RNAs within the RdDM pathway.