A Two-Hit Trigger for siRNA Biogenesis in Plants

Abstract

In Arabidopsis, microRNA-directed cleavage can define one end of RNAs that then generate phased siRNAs. However, most miRNA-targeted RNAs do not spawn siRNAs, suggesting the existence of additional determinants within those that do. We find that in moss, phased siRNAs arise from regions flanked by dual miR390 cleavage sites. AtTAS3, an siRNA locus important for development and conserved among higher plants, also has dual miR390 complementary sites. Both sites bind miR390 in vitro and are functionally required in Arabidopsis, but cleavage is undetectable at the 5' site--demonstrating that noncleavable sites can be functional in plants. Phased siRNAs also emanate from the bounded regions of every Arabidopsis gene with two known microRNA/siRNA complementary sites, but only rarely from genes with single sites. Therefore, two "hits,"--often, but not always, two cleavage events--constitute a conserved trigger for siRNA biogenesis, a finding with implications for recognition and silencing of aberrant RNA.

A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis

“…The TAS3a-c transcripts (pri-TAS3a-c) that require two miR390 target sites for tasiRNA production have some short ORFs, but no . 30-nt ORFs that encompass either miR390 target site are found (Axtell et al, 2006;Howell et al, 2007;Montgomery et al, 2008a). The fact that tasiRNAs are produced even without ORFs that encompass miRNA recognition sites is consistent with our results of mutational analysis for TAS2 ORF3 and indicates that these ORFs are not absolutely required but facilitate tasiRNA production.…”

“…These modifications allow the export of transcripts from the nucleus to the cytoplasm where pri-TASs are cleaved by AGO1-RISCs containing 22-nucleotide (nt) miRNAs (e.g. microRNA173 [miR173] in Arabidopsis [Arabidopsis thaliana]) or AGO7-RISCs containing microRNA390 (miR390; Axtell et al, 2006;Chen et al, 2010;Cuperus et al, 2010;Felippes and Weigel, 2009;Montgomery et al, 2008aMontgomery et al, , 2008b. The cleavage results in poly(A)-deficient 59 fragments and uncapped 39 fragments, which are associated with the RISC and SUPPRESSOR OF GENE SILENCING3 (SGS3) and protected from degradation (Yoshikawa et al, 2005.…”

Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry

“…The phasiRNAs, including one tasiARF, are produced downstream of both cleavage sites. The demonstrated cleavage at both 39 and 59 sites of fve-TAS3S by the PARE data argues against an absolute requirement for a noncleavable 59 site in the two-hit mode of tasiARF biogenesis (Axtell et al, 2006;Allen and Howell, 2010). miR828 is conserved in eudicots (Cuperus et al, 2011); although it was not identified in our list of known or conserved miRNAs due to its extremely low expression level, its cleavage of fve-TAS4 and tasiRNA production were confirmed by the PARE data and phasing analyses, respectively (Fig.…”

Short Silencing RNA: The Dark Matter of Genetics?

“…It could also be, as indicated in a very recent report, that a "normal" RNA with two sRNA target sites could also become a source of secondary sRNAs and, consequently, a node (Axtell et al 2006). RNAs with aberrancy at both ends might also generate sRNAs in the absence of target- Figure 4.…”