Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA-dominated PHAS loci.

Seth Polydore,Michael J. Axtell,Alice Lunardon

doi:10.1002/pld3.101

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 (PHAS) are well represented in all land plants to date. In contrast, 24 nt‐dominated PHAS 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 PHAS loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt‐dominated PHAS loci also exist in Arabidopsis thaliana. We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt‐dominated PHAS loci using multiple PHAS‐detecting algorithms. We also find that MIR2275 is not found in A. thaliana, and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used PHAS‐detecting algorithms which may lead to false positives when trying to annotate new PHAS, especially 24 nt‐dominated loci.

Highlights

Small RNAs regulate key physiological functions in land plants, ranging from organogenesis (Boualem et al, 2008, 2008; Kutter et al, 2007; Laufs et al, 2004; Williams et al, 2005) to gametogenesis (Grant-Downton et al, 2009)
The AGO protein that loads these phasiRNAs is unknown; in maize, AGO18b expression levels match those of the 24 nt-dominated PHAS loci quite closely and is the most likely candidate to load 24 nt phasiRNAs (Komiya et al, 2014; Zhang et al, 2015). The targets of these 24 nt phasiRNAs are unknown, but they are apparently necessary for proper male gametogenesis (Ono et al, 2018). 24 nt-dominated PHAS loci were described in the non-grass monocots asparagus, lily, and daylily (Kakrana et al, 2018). These phasiRNAs are produced from processing of inverted repeat (IR) RNAs, instead of the double-stranded RNA precursors observed in rice and maize (Kakrana et al, 2018)
We interrogated small RNA libraries for these species to see if a mature miR2275 homolog was expressed

Summary

Introduction

Small RNAs regulate key physiological functions in land plants, ranging from organogenesis (Boualem et al, 2008, 2008; Kutter et al, 2007; Laufs et al, 2004; Williams et al, 2005) to gametogenesis (Grant-Downton et al, 2009). Much like 21 nt-dominated PHAS, the biogenesis of these 24 nt-dominated PHAS loci begins with the Pol II-dependent transcription of a single-stranded RNA precursor which is targeted by miR2275 and hydrolyzed. The resulting RNA transcript is converted into a double-stranded RNA molecule by RDR6 (Zhai et al, 2015) These phasiRNA precursors are hydrolyzed by DCL5 (a DCL3 homolog sometimes called DCL3b) to produce 24 nt phasiRNAs 73 (Fei et al, 2013). 24 nt-dominated PHAS loci were described in the non-grass monocots asparagus, lily, and daylily (Kakrana et al, 2018) These phasiRNAs are produced from processing of inverted repeat (IR) RNAs, instead of the double-stranded RNA precursors observed in rice and maize (Kakrana et al, 2018). Our analysis shows that existing phasing score algorithms to detect novel PHAS loci can lead to false positives

Results

Discussion

Materials and Methods

486 Literature cited