Epigenetic and biochemical determinants of nuclear siRNA biogenesis in Arabidopsis – Epi_siRNA_biogen

Cells have RNA-directed mechanisms to destroy viruses, silence transposons and regulate genes. Such “RNA silencing” systems rely upon selective base pairing of small interfering RNAs (siRNAs) to complementary RNA targets. RNA silencing targets specific mRNAs for translational inhibition, cleaves transcripts, or silences genomic loci transcriptionally. In terrestrial plants, multiple biogenesis pathways have evolved to process double-stranded RNA (dsRNA) into functionally distinct siRNAs. Failure to channel dsRNA via an appropriate pathway can disrupt organ patterning or render cells vulnerable to deleterious mutations. siRNA biogenesis begins with the synthesis of precursors at particular genomic source loci. This project focuses on RNA polymerase IV (Pol IV) and its partner RDR2, which synthesize dsRNA substrates that are processed by Dicer-like 3 (DCL3) into siRNAs that guide methylation and silence transposons. Safeguards on siRNA pathway integrity will be investigated, including the epigenetic marks that recruit Pol IV, the mechanism of Pol IV-RDR2 physical coupling, and selective functions of non-catalytic domains in enzymes of the Pol IV pathway. The project will use high-resolution chromatin-immunoprecipitation (ChIP-nexus) to study Pol IV recruitment genome-wide and a modified CRISPR/Cas9 system to deliver Pol IV to genomic loci of choice. With these approaches, Pol IV recognition of new transposon insertions and the initiation of RNA-directed DNA methylation will be dissected with exceptional precision. We present preliminary data for pol IV missense mutations and propose innovative strategies for isolating novel mutations in non-catalytic domains of RDR2. A genetic suppressor screen using a pol IV missense line aims to discover regulators of Pol IV activity. This research will elucidate conserved structure-function relationships that allow paralogous enzyme consortia to operate distinct siRNA pathways in the same cell. The mechanistic understanding of Pol IV targeting and siRNA biogenesis gained from the work will help scientists make precise adjustments to genetic traits and better harness the RNA silencing repertoire in plants.