TRACKING SRNAS WITH UNTEMPLATED NUCLEOTIDES IN THE RNA DIRECTED DNA METHYLATION PATHWAY

Abstract

The production of 23-nt sRNAs within the RNA-directed DNA Methylation (RdDM) pathway is a process that is assumed to depend on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) but which has rarely been studied directly. Recent studies suggest that RDR2 leaves an untemplated nucleotide at the 3’ end of its transcription product via terminal transferase activity (TTA). In this study, I sought to identify these RDR2-produced 23-nt sRNAs with terminal 3’ untemplated nucleotides (23* sRNAs) in sRNA sequencing data from both Brassica rapa and Arabidopsis thaliana and quantitatively characterize these reads relative to perfectly-mapping 23-nt and 24-nt reads at the whole-plant, tissue, and locus level. While I was able to identify 23* reads as a compositionally distinct subset of 23-nt reads, I found that their production frequency is much lower than that of perfectly-mapping 23-nt and 24-nt reads. However, 23* reads also frequently map to the same genomic locations as perfectly-mapping 23-nt and 24-nt reads in an RDR2-dependent manner, indicating that 23* reads are produced as part of RdDM. I present a series of models to explain potential mechanisms of 23-nt sRNA production within the RdDM pathway.