Reconstituting plant miRNA biogenesis

Abstract

The genomes of higher eukaryotes encode not only proteins but also diverse noncoding RNAs, particularly small (20- to 30-nt) regulatory RNAs (1–3). The small RNAs include microRNAs (miRNAs), siRNAs, and piwi-interacting RNAs (piRNAs) (4, 5). These small RNAs repress gene expression at the transcriptional or posttranscriptional levels and have critical functions in genome defense, growth, development, diseases, and stress responses (1, 3, 6–8). Small RNAs are classified largely on the basis of their biogenesis requirements. miRNAs arise from single-stranded primary miRNA transcripts (pri-miRNAs) that can form imperfect stem–loop structures (6) (Fig. 1). In animals, pri-miRNAs are processed in the nucleus into shorter hairpin RNAs of ≈65 nt (premiRNAs) by the microprocessor complex containing the RNaseIII enzyme Drosha and its cofactor DGCR8/Pasha, a dsRNA-binding protein (5, 9). The premiRNA is then exported to the cytoplasm, where it is further processed by another RNaseIII enzyme, Dicer, to release an ≈22-nt miRNA/miRNA* duplex (5, 9). Dicer function also requires a dsRNA-binding protein, TRBP, as a cofactor. The miRNA is loaded into the effector complex, known as RISC, to direct complementary or partially complementary mRNAs for cleavage or translational repression (5, 6). In plants, the two-step processing of pri-miRNAs into mature miRNAs occurs entirely in the nucleus and is carried out by a single RNaseIII enzyme, DCL1 (Dicer-like 1) (6). In addition to DCL1, genetic analysis revealed that HYL1, a dsRNA-binding protein, and SE, a C2H2-type zinc finger, are also required for processing pri-miRNAs and for accumulation of mature miRNAs (10–12) (Fig. 1). However, whether DCL1 alone is active in processing pri-miRNAs into miRNAs and how HYL1 and SE may function in the processing steps are not known. In this issue of PNAS, Dong et al. (13) reconstituted the processing of pri-miRNAs in vitro by using recombinant proteins and thereby provided much-needed biochemical data to explain the genetic roles of DCL1, HYL1, and SE in miRNA biogenesis in plants.