Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA‐dominated <i>PHAS</i> loci

Abstract

Small RNAs regulate key physiological functions in land plants. Small RNAs can be divided into two categories: microRNAs (miRNAs) and short interfering RNAs (siRNAs); siRNAs are further subdivided into transposon/repetitive region-localized heterochromatic siRNAs and phased siRNAs (phasiRNAs). PhasiRNAs are produced from the miRNA-mediated cleavage of a Pol II RNA transcript; the miRNA cleavage site provides a defined starting point from which phasiRNAs are produced in a distinctly phased pattern. 21-22 nucleotide (nt)-dominated phasiRNA-producing loci (<i>PHAS</i>) are well represented in all land plants to date. In contrast, 24 nt-dominated <i>PHAS</i> loci are known to be encoded only in monocots and are generally restricted to male reproductive tissues. Currently, only one miRNA (miR2275) is known to trigger the production of these 24 nt-dominated <i>PHAS</i> loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated <i>PHAS</i> loci also exist in <i>Arabidopsis thaliana</i>. We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt-dominated <i>PHAS</i> loci using multiple <i>PHAS-</i>detecting algorithms. We also find that <i>MIR2275</i> is not found in <i>A. thaliana</i>, and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used <i>PHAS-</i>detecting algorithms which may lead to false positives when trying to annotate new <i>PHAS</i>, especially 24 nt-dominated loci.