Identification and characterisation of MORC6 as a component of the RNA-directed DNA methylation pathway in Arabidopsis thaliana

RNA silencing pathways control the expression of genes and other DNA loci by the action of small RNA molecules and are found in many eukaryotes. In plants there are a number of RNA silencing pathways, of which RNA-directed DNA methylation (RdDM) is one. In this pathway the small RNA molecules direct DNA methylation, resulting in the down regulation of expression of the target locus. In terms of the mechanism of the pathway it is mostly well characterised but several gaps exist in our knowledge. These relate to its initiation, where it is not known how RdDM targets the correct locus; methylation, where it is unclear how the action of small RNAs triggers methylation; and chromatin modification, where it is unclear how methylated DNA is converted into higher order chromatin modification. These gaps in the pathway raised the possibility of the involvement of novel proteins and so this project aimed to identify and characterise mutants in these proteins.

Screening of a library of putative RdDM mutants in Arabidopsis thaliana identified three alleles in MORC6, which encodes a GHKL ATPase containing protein associated with RdDM that is thought to form higher order chromatin in response to DNA methylation. Analysis of the three alleles revealed that morc6 mutants have no effect on siRNA production but at certain loci do have an effect on DNA methylation and so would suggest that MORC6 is also involved in the DNA methylation process in RdDM at specific loci. It was also shown that silencing by RdDM can still occur in morc6 mutants in a limited capacity and that this silencing is stochastic and cell autonomous in nature. These findings point to MORC6 also having further roles in RdDM other than higher order chromatin modification and so increase our understanding of the mechanism of RdDM.