Sequencing and characterization of non-coding small RNAs controlling development in Arabidopsis thaliana roots

Abstract

Small noncoding RNAs (ncRNAs) are key regulators of plant development through modulation of the processing, stability and translation of larger RNAs. I generated small RNA datasets comprising over 200 million aligned Illumina sequence reads covering all major cell types of the root as well as four distinct developmental zones. These data were analyzed for three major types of small RNAs, namely microRNAs (miRNAs), repeat associated small interfering RNAs (ra-siRNAs), and trans-acting siRNAs (ta-siRNAs). 133 of the 243 known miRNAs were found to be expressed in the root, and most showed tissue- or zone-specific expression patterns. My collaborators and I identified 70 new high-confidence miRNAs, and knockdown of three of the newly identified miRNAs resulted in altered root growth phenotypes. Ra-siRNAs specify methylation by the RNA directed DNA methylation (RdRM) pathway, requiring the generation of additional methylation datasets. Preliminary analysis shows cell-type specific methylation patterns that correlate with small RNA and mRNA expression. Analysis of ta-siRNAs revealed new ta-siRNA generating loci, and a novel triggering miRNA for TAS1 loci. In summary, our study demonstrates the power of isolating individual cell types and developmental zones in combination with deep sequencing and computational analyses to obtain detailed profiles of ncRNAs, as well as to significantly extend the compendium of known functional RNAs.