Transcription from an upstream promoter controls methylation signaling from an inverted repeat of endogenous genes inArabidopsis

Abstract

In plants, replication of RNAviruses and RNAfrom highly transcribed transgenes can trigger DNA methylation. These systems accumulate diced small RNA(smRNA) products of double-stranded RNA(dsRNA) precursors, but it is not known which RNAspecies directs methylation. The methylatedPAItryptophan biosynthetic genes inArabidopsisallow the study of methylation signals for endogenous genes with lower expression levels. ThePAIgenes are arranged as a tandem inverted repeat plus two singlet genes at unlinked loci. Here we show that the predominantPAItranscript initiates at a novel unmethylated promoter that lies upstream of one of the inverted repeatPAIgenes. Suppressed transcription from the upstream promoter using transgene-directed silencing reduces methylation on the singletPAIgenes, but not on the inverted repeat, consistent with an RNAmethylation signal. RNAgel blots detect normalPAItranscripts and dsRNA read-through species, but not diced smRNAs, suggesting that either precursor dsRNAs or subdetectable levels of smRNAs, below the threshold to effectively degradePAItranscripts, serve as thePAImethylation signal. Thus, the lower expression endogenous gene system allows dissection of a RNA-directed methylation pathway distinct from RNA degradation pathways.