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

Abstract

Small RNA s regulate key physiological functions in land plants. Small RNA s can be divided into two categories: micro RNA s (mi RNA s) and short interfering RNA s (si RNA s); si RNA s are further subdivided into transposon/repetitive region‐localized heterochromatic si RNA s and phased si RNA s (phasi RNA s). Phasi RNA s are produced from the mi RNA ‐mediated cleavage of a Pol II RNA transcript; the mi RNA cleavage site provides a defined starting point from which phasi RNA s are produced in a distinctly phased pattern. 21–22 nucleotide (nt)‐dominated phasi RNA ‐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 mi RNA (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 si RNA s were consistently misidentified as 24 nt‐dominated PHAS loci using multiple PHAS ‐ detecting algorithms. We also find that MIR 2275 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.

Abundant expression of maternal siRNAs is a conserved feature of seed development

“…The biological role of these phased siRNAs is unknown, but it is striking that they are produced from sporophytic tissue surrounding the developing gamete, the same pattern we see from siren siRNAs. We detect elevated phasing scores at siren loci; however highly-expressed siRNA loci can be misidentified by common phasing algorithms (Polydore et al, 2018). Because Pol IV produces transcripts only slightly longer than a single siRNA (Blevins et al, 2015;Zhai et al, 2015), it is unlikely that 24-nt siren RNAs are phased.…”

Integrated annotations and analyses of small RNA–producing loci from 47 diverse plants

“…By default, ShortStack assigns a phasing score to the sRNA loci based on the algorithm described in Guo et al (2015). However, an accurate annotation of the phasing would require a more complex study to avoid false positives that may be produced by the commonly used phasing-detecting algorithms (Polydore et al 2018). Therefore, we did not further analyze the phasing of the sRNA loci in this analysis.…”

The floral development of the allotetraploid Coffea arabica L. correlates with a small RNA dynamic reprogramming

“…These loci exhibit significant enrichment of mapped reads in reproductive organs, particularly anthers (Pokhrel et al., 2021; Pokhrel & Meyers, 2022; Xia et al., 2019). Because several phased siRNA annotation methods can frequently mistake heterochromatic siRNAs with 24 ‐PHAS loci (Polydore et al., 2018), we manually evaluated all putative PHAS loci with IGV. Doing so, we were able to identify 189 24‐ PHAS loci (Figure 1—depicted in the second concentric cycle starting from the center, marked with orange dots; Table S3).…”