microRNAs (miRNAs) play crucial roles in numerous developmental and metabolic processes in plants and animals. The steady-state levels of miRNAs need to be properly controlled to ensure normal development. While the framework of miRNA biogenesis is established, factors involved in miRNA degradation remain unknown. Here, we show that a family of exoribonucleases encoded by the SMALL RNA DEGRADING NUCLEASE (SDN) genes degrades mature miRNAs in Arabidopsis. SDN1 acts specifically on single-stranded miRNAs in vitro, and is sensitive to the 2'-O-methyl modification on the 3' terminal ribose of miRNAs. Simultaneous knockdown of three SDN genes in vivo results in elevated miRNA levels and pleiotropic developmental defects. Therefore, we have uncovered the enzymes that degrade miRNAs and demonstrated that miRNA turnover is crucial for plant development.

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs), control gene expression and epigenetic regulation. Although the roles of miRNAs and siRNAs have been extensively studied, their expression diversity and evolution in closely related species and interspecific hybrids are poorly understood. Here, we show comprehensive analyses of miRNA expression and siRNA distributions in two closely related species Arabidopsis thaliana and Arabidopsis arenosa, a natural allotetraploid Arabidopsis suecica, and two resynthesized allotetraploid lines (F 1 and F7) derived from A. thaliana and A. arenosa. We found that repeat-and transposon-associated siRNAs were highly divergent between A. thaliana and A. arenosa. A. thaliana siRNA populations underwent rapid changes in F 1 but were stably maintained in F7 and A. suecica. The correlation between siRNAs and nonadditive gene expression in allopolyploids is insignificant. In contrast, miRNA and tasiRNA sequences were conserved between species, but their expression patterns were highly variable between the allotetraploids and their progenitors. Many miRNAs tested were nonadditively expressed (deviating from the midparent value, MPV) in the allotetraploids and triggered unequal degradation of A. thaliana or A. arenosa targets. The data suggest that small RNAs produced during interspecific hybridization or polyploidization serve as a buffer against the genomic shock in interspecific hybrids and allopolyploids: Stable inheritance of repeat-associated siRNAs maintains chromatin and genome stability, whereas expression variation of miRNAs leads to changes in gene expression, growth vigor, and adaptation. expression regulation ͉ microRNAs ͉ polyploidy ͉ hybrid vigor

Proteins containing the forkhead-associated domain (FHA) are known to act in biological processes such as DNA damage repair, protein degradation, and signal transduction. Here we report that DAWDLE (DDL), an FHA domain-containing protein in Arabidopsis, acts in the biogenesis of miRNAs and endogenous siRNAs. Unlike mutants of genes known to participate in the processing of miRNA precursors, such as dcl1, hyponastic leaves1, and serrate, ddl mutants show reduced levels of pri-miRNAs as well as mature miRNAs. Promoter activity of MIR genes, however, is not affected by ddl mutations. DDL is an RNA binding protein and is able to interact with DCL1. In addition, we found that SNIP1, the human homolog of DDL, is involved in miRNA biogenesis and interacts with Drosha. Therefore, we uncovered an evolutionarily conserved factor in miRNA biogenesis. We propose that DDL participates in miRNA biogenesis by facilitating DCL1 to access or recognize pri-miRNAs.class of sequence-specific repressors of gene expression in eukaryotes is 20-to 24-nt small RNAs, which include miRNAs and siRNAs. miRNAs are processed from stem-loop precursor RNAs, called pri-miRNAs. In animals, pri-miRNAs are processed in the nucleus by Drosha to form pre-miRNAs, which are exported to the cytoplasm by exportin 5 and further processed by Dicer to produce mature miRNAs (reviewed in ref. 1). In Arabidopsis, mature miRNAs are produced through two processing steps (primiRNAs to pre-miRNAs and pre-miRNAs to miRNAs) in the nucleus by DCL1 with the assistance of HYL1 and SERRATE (reviewed in ref. 2). After processing, miRNAs are 2Ј-O-methylated by HEN1 (3). siRNAs are produced from long, double-stranded RNAs. Plants contain several classes of endogenous siRNAs, such as transacting siRNAs (ta-siRNAs), natural antisense siRNAs (nat-siRNAs), and siRNAs from endogenous repeat sequences and transposons (reviewed in ref. 4).The forkhead-associated (FHA) domain is an 80-to 100-aa module that is thought to recognize phosphothreonine-containing motifs and mediate protein-protein interactions in prokaryotes and eukaryotes (reviewed in ref. 5). DAWDLE (DDL) is a nuclearlocalized FHA domain-containing protein in Arabidopsis (6). DDL appears to act in multiple developmental processes such as growth, fertility, and root, shoot, and floral morphogenesis (6).Smad nuclear interacting protein 1 (SNIP1) is a human FHA domain-containing protein that functions as an inhibitor of TGF-␤ and NF-B signaling pathways by competing with the TGF-␤ signaling protein Smad4 and the NF-B transcription factor p65/ RelA for binding to the transcriptional coactivator p300 (7,8). Recently, Fujii et al. (9) reported that SNIP1 interacts with the transcription factor/oncoprotein c-Myc and enhances its activity by bridging its interaction with p300.Here we report that DDL is required for the accumulation of miRNAs and endogenous siRNAs in Arabidopsis. Its affinity for RNA, its potential association with DCL1, and the reduction in pri-miRNA levels in ddl loss-of-function mutants suggest that DDL is...

SUMMARY microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) impact numerous biological processes in eukaryotes. In addition to biogenesis, turnover contributes to the steady-state levels of small RNAs. One major factor that stabilizes miRNAs and siRNAs in plants as well as siRNAs and piRNAs in animals is 2′;-O-methylation on the 3′; terminal ribose by the methyltransferase HUA ENHANCER1 (HEN1) [1–6]. Genetic studies with Arabidopsis, Drosophila and zebrafish hen1 mutants show that 2′-O-methylation protects small RNAs from 3′-to-5′ truncation and 3′ uridylation, the addition of non-templated nucleotides, predominantly uridine [2, 7, 8]. Uridylation is a widespread phenomenon that is not restricted to small RNAs in hen1 mutants, and is often associated with their reduced accumulation ([7, 9, 10]; reviewed in [11]). The enzymes responsible for 3′ uridylation of small RNAs when they lack methylation in plants or animals have remained elusive. Here, we identify the Arabidopsis HEN1 SUPPRESSOR1 (HESO1) gene as responsible for small RNA uridylation in hen1 mutants. HESO1 exhibits terminal nucleotidyl transferase activity, prefers uridine as the substrate nucleotide, and is completely inhibited by 2′-O-methylation. We show that uridylation leads to miRNA degradation, and the degradation is most likely through an enzyme that is distinct from that causing the 3′ truncation in hen1 mutants.

Posttranscriptional gene silencing (PTGS) in plants isPosttranscriptional gene silencing (PTGS) involves the degradation of viral and cellular mRNAs in a homology-dependent manner, and it is conserved in diverse eukaryotes (15,25). In plants, PTGS functions as a natural antiviral defense because plant viruses are both initiators and targets of PTGS (47). PTGS was first discovered in plants (30); however, a mechanistically similar phenomenon was later described in other organisms: it is called quelling in fungi (8) and RNA interference in Caenorhabditis elegans (11) and in Drosophila melanogaster (16). Recent studies at the molecular level revealed that all of these can be considered to be manifestations of an RNA-targeting pathway. Even though the mechanism by which a virus infection triggers PTGS in plants is not fully understood, double-stranded RNA (dsRNA) has been found to be a strong inducer of PTGS (57). Such a form is produced during replication of an RNA virus or conversion of aberrant single-stranded RNAs into dsRNA in the cell by host-encoded RNA-directed RNA polymerase. These dsRNAs are first processed into 21-to 26-nt short interfering RNAs (siRNAs) by an RNase DICER enzyme and subsequently serve as guides by forming an active multicomplex RNA-induced silencing complex, which cleaves homologous RNA molecules (5). In plants, gene silencing generates an unknown mobile signal that can trigger PTGS in distant tissues and across a graft union (32).In recent years, RNA silencing-inhibiting proteins that counter antiviral RNA silencing have been identified in several plant viruses (47) and in an insect virus (25). These identified silencing suppressor proteins may act at different steps in the PTGS pathway. Three distinct phases have been identified in the RNA-silencing process: initiation, maintenance, and systemic signaling. Thus, (i) the potyvirus helper component proteinase (HC-Pro) interferes with the initiation and maintenance of silencing at a step coincident with or upstream of siRNA production, because it did not prevent the silencing signal from becoming systemic (1,22,26); (ii) the 2b protein of Cucumber mosaic virus (CMV) prevents the initiation of PTGS in newly emerging tissues by inhibiting long-range PTGS-signaling activity (6, 13); and (iii) p25 of Potato virus X (PVX) suppresses the production or accumulation of the mobile silencing signal (54). Recently, the p19 protein of tombusviruses was implicated in inhibiting RNA silencing by physically interacting with siRNAs and thus providing another mechanism to interfere with RNA silencing (43). In geminiviruses, AC2, encoding the transcriptional activator protein (TrAP) of the Kenyan strain of African cassava mosaic virus (ACMV- [KE]), and the product of C2, a positional homologue of AC2 in the monopartite Tomato yellow leaf curl China virus (TYLCCNV) have both been identified as suppressors of PTGS (52,55).In nature, mixed viral infections occur in the same plant,