MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1–HYL1–SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis . Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA–HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.

Significance: CDC5, a DNA binding protein, regulates the growth and immunity of Arabidopsis , however, its functional mechanism remains to be identified. This research reveals that CDC5 acts as a positive transcription factor and a component of the DICER–LIKE1 complex to regulate microRNA (miRNA) accumulation. By identifying a unique regulator and its activities in miRNA biogenesis, this study provides insight on the regulation of miRNA levels. Because CDC5 homologs are present in other organisms, including animals, this study may have a broad effect on the miRNA biogenesis field.

In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. Significance This study, for the first time to the authors9 knowledge, establishes HUA1 enhancer 1 (HEN1) suppressor 1 as a 5′ fragment uridyl transferase and shows that uridylation triggers the degradation of 5′ fragments. This study also demonstrates that HEN1 suppressor 1 interacts with Argonaute (AGO1) and is able to act on microRNA substrates in the AGO1 complex. Furthermore, this study reveals that methylation protects microRNAs from AGO1-associated uridylation activity in plants. Because methylation and uridylation are conserved processes in small RNA pathways in plants and animals, this study may have a broad effect in related fields.

MicroRNAs (miRNAs) are essential non-coding riboregulators of gene expression in plants and animals. In plants, miRNAs guide their effector protein named ARGONAUTE (AGO) to find target RNAs for gene silencing through target RNA cleavage or translational inhibition. miRNAs are derived from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by the DNA-dependent RNA polymerase II. In plants, an RNase III enzyme DICER-LIKE1-containing complex processes pri-miRNAs in the nucleus into miRNAs. To ensure proper function of miRNAs, plants use multiple mechanisms to control miRNA accumulation. On one hand, pri-miRNA levels are controlled through transcription and stability. On the other hand, the activities of the DCL1 complex are regulated by many protein factors at transcriptional, post-transcriptional and post-translational levels. Notably, recent studies reveal that pri-miRNA structure/sequence features and modifications also play important roles in miRNA biogenesis. In this review, we summarize recent progresses on the mechanisms regulating miRNA biogenesis.