DICER-LIKE3 Activity in Physcomitrella patens DICER-LIKE4 Mutants Causes Severe Developmental Dysfunction and Sterility

Abstract

Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci.

Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants

“…Most DCL-Derived Small RNA Loci Produce Mixtures of 23-to 24-Nucleotide Small RNAs in P. patens Several previous studies have annotated P. patens miRNAs and endogenous siRNAs using small RNA-seq (Arazi et al, 2005;Axtell et al, 2006;Fattash et al, 2007;Cho et al, 2008;Arif et al, 2012). However, these previous small RNA-seq efforts all had low sequencing depth by current standards (<0.5 million mapped reads per library in all cases).…”

Phased, Secondary, Small Interfering RNAs in Posttranscriptional Regulatory Networks

“…In Physcomitrella patens, miR390, TAS3a, and the resulting tasi-ARFs have all been described (Axtell et al, 2006;Talmor-Neiman et al, 2006), as well as additional TAS loci, some of which are not conserved with Arabidopsis (Arif et al, 2012). The P. patens TAS6 is a two-hit locus like TAS3 but is targeted by different conserved miRNAs, and it has important roles in development, including bud formation .…”

“…In addition, the "TAS" name has been given only to noncoding transcripts with no function other than to give rise to secondary siRNAs. Recent work has described the TAS6 locus (Arif et al, 2012) and many TAS-like (TASL) loci (R. , as well as an as yet unnamed TAS-like ncRNA locus (Zhai et al, 2011), indicating that additional ncRNA-derived TAS loci will continue to be described and named, some of which may be lineage specific. TAS5 has also been described ), but we believe it is inappropriately named, as it appears to be an incorrectly annotated protein coding (NB-LRR) transcript.…”

Novel DICER-LIKE1 siRNAs Bypass the Requirement for DICER-LIKE4 in Maize Development

“…hierarchically acting DCL enzymes (Arif et al, 2012). Thus, it is noteworthy that in maize, a 21-nucleotide class of tasiR-ARFs remains, even in dcl4-2 mutants.…”

“…Hierarchy among DCL proteins has been noted in other plants. For example, in Arabidopsis, tomato, and moss, mutation of DCL4 allows the processing of TAS precursor transcripts into 22-to 24-nucleotide siRNAs by DCL2 and/or DCL3 Arif et al, 2012;Yifhar et al, 2012). However, the increased length of these ta-siRNAs offsets their normal phasing pattern such that functional tasiR-ARFs are no longer produced.…”