Novel miR390‐Dependent Transacting siRNA Precursors in Plants Revealed by a PCR‐Based Experimental Approach and Database Analysis

Abstract

TAS loci in plant genomes encode transacting small interfering RNAs (ta-siRNAs) that regulate expression of a number of genes. The function of TAS3 precursor in Arabidopsis thaliana is controlled by two miR390 target sites flanking two ta-siARF sequences targeting mRNAs of ARF transcription factors. Cleavage of the 3′-miR390-site initiates ta-siRNAs biogenesis. Here we describe the new method for identification of plant ta-siRNA precursors based on PCR with oligodeoxyribonucleotide primers mimicking miR390. The method was found to be efficient for dicotiledonous plants, cycads, and mosses. Based on sequences of amplified loci and a database analysis, a novel type of miR390-dependent TAS sequences was identified in dicots. These TAS loci are characterized by a smaller distance between miR390 sites compared to TAS3, a single copy of ta-siARF, and a sequence conservation pattern pointing to the possibility that processing of novel TAS-like locus is initiated by cleavage of the 5′-terminal miR390 target site.

Molecular Diversity of miR390-Guided Transacting siRNA Precursor Genes in Lower Land Plants: Experimental Approach and Bioinformatics Analysis

“…To apply the approach based on PCR amplification [15] to TAS3-like loci of lower land plants, we designed degenerated primers BryoTAS-P and Bryo TAS-M Figure 1: Analysis of PCR products in 1.5% agarose gel. Amplification of genomic DNA sequences flanked by miR390 sites.…”

“…For amplification, the Physcomitrella TAS3-specific primers [15] were used: a forward primer Bryo TAS-P 5 -GRCGYTAYCCTCYTGAGCT-3 and reverse primer Bryo TAS-M 5 -TAGCTCAGGAGTGATA(G/T)A(C/A)AA-3 . For PCR, 25-35 cycles were used for amplification with a melting temperature of 95…”

“…The method was found to be efficient for dicotyledonous plants, cycads, conifers, and mosses [15]. In this study, PCR-based approach was used as a phylogenetic profiling tool to probe genomic DNA samples derived from Selaginella kraussiana (Lycopodiopsida) and 13 bryophyta species from classes Bryopsida (subclasses Bryidae and Dicranidae) and Sphagnopsida (Sphagnum squarrosum and Sphagnum girgensohnii).…”

Phased, Secondary, Small Interfering RNAs in Posttranscriptional Regulatory Networks

“…In Arabidopsis, only the 39 miRNA target site in the TAS3 transcript is cleaved, as in other flowering plants, while both miR390 complementary sites in moss and pine showed cleavage ( Figure 2B) (Axtell et al, 2006). A shorter variant of TAS3 than that found in Arabidopsis is also conserved in many eudicots and is found alongside the canonical TAS3 locus; this variant has cleavable target sites at both 59 and 39 positions and includes only a single tasiARF (Figure 2A) (Krasnikova et al, 2009;Xia et al, 2012). A recent study of phasiRNA trigger evolution (e.g., miRNAs) across a broad range of plant (A) As the first step in secondary siRNA biogenesis, mRNA targets are cleaved by a miRNA.…”

“…The two-hit model provided the first mechanistic insights into the process for tasiRNA biogenesis, describing TAS3 as the prototypical two-hit locus (Axtell et al, 2006). Early experimental examination of the two TAS3 target sites demonstrated that miR390 has an unusual association with AGO7 that is important for tasiRNA biogenesis and that the 59 proximal miR390 target site must not be cleaved (Montgomery et al, 2008a), although outside of Arabidopsis, TAS3 variants may be cleaved at the 59 position (Axtell et al, 2006;Krasnikova et al, 2009;Xia et al, 2012). However, more recent data from other plant genomes describe two-hit loci for which the miRNA triggers are believed to be AGO1 associated and for which the 59 proximal site may be cleaved.…”