Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body

Abstract

Small regulatory microRNAs (miRNAs) are encoded in long precursors and are released from them during processing by cleavage within partially duplexed stem-loop structures. In the present work we investigated the role of the Arabidopsis nuclear RNA-binding protein HYL1 and the nuclear RNase III enzyme DCL1 in processing of primary miRNA (pri-miR171a). The miR171a gene is complex, with multiple transcription start sites, as well as alternative splicing of exons and alternative polyadenylation sites. Both HYL1 and DCL1 proteins are required for processing of the major pri-miR171a, spliced and polyadenylated forms of which accumulate in plants homozygous for mutations in either gene, but not in wild-type plants. In transiently transfected Arabidopsis protoplasts, HYL1-mCherry and YFP-DCL1 fusion proteins colocalize to small nuclear bodies similar to Cajal bodies but lacking the Cajal body marker Atcoilin. The HYL1 protein coimmunoprecipitates with miR171a and miR159a precursors, indicating that it is an integral component of the precursor processing machinery. Thus, the distinct HYL1-and DCL1-containing nuclear bodies may be miRNA precursor processing sites. Alternatively, they may be assembly and storage sites for the miRNA precursor processing machinery.small regulatory RNA ͉ precursor ͉ plant M icroRNAs (miRNAs) are small regulatory RNAs encoded in the genomes of both plants and animals. The miRNA sequence is embedded within a much larger primary miRNA (pri-miRNA) transcript and cleaved out in two steps. The first cleavage generates a stem-loop intermediate, termed the precursor miRNA (pre-miRNA), and the second cleavage releases the miRNA duplex, one strand of which is used as the regulatory effector (1). Mammals use a single class III RNase III, Dicer, to generate both miRNAs and siRNAs, whereas plants have multiple specialized Dicer-like (DCL) enzymes. Among the four Arabidopsis DCLs, DCL1 participates in miRNA biogenesis, whereas the others are involved in various aspects of small RNA-mediated gene silencing (1, 2). In animal cells, pri-miRNAs are processed to pre-miRNAs by the RNase III Drosha in the nucleus, whereas Dicer cleaves the pre-miRNAs in the cytoplasm. Both cleavages are thought to be carried out in the nucleus by DCL1 in Arabidopsis (3). Double-stranded RNA-binding proteins (dsRBPs) participate in both the biogenesis and function of small regulatory RNAs, including miRNA and siRNA. In animals, RNase III family enzymes almost always pair with a dsRBP in miRNA precursor and dsRNA processing and are components of the RNA-induced silencing complex (1). Human DGCR8, known as Pasha in Caenorhabditis elegans and Drosophila, is the dsRBP partner of Drosha in the nuclear Microprocessor complex that cleaves pri-miRNA to premiRNA (4, 5). The Arabidopsis HYL1 dsRBP, identified through characterization of an insertion mutant designated hyponastic leaves 1 (hyl1), is required for miRNA biogenesis (6-8). Although little is known about its mechanism of action, it has been reported that HYL1 can bind to DCL1 and ...

Alternate approaches to repress endogenous microRNA activity inArabidopsis thaliana

“…Recent studies have localized the miRNA biogenesis machinery proteins DCL1, DRB1 and SE to the nucleus specialized D-bodies. 10,11 In addition, HEN1 and AGO1 also show some localization to the nucleus and to D-bodies. 11 In plants, the Slicer activity of AGO1 specifically co-elutes with a small protein complex, 19 to suggest that miRNA-loaded RISC may consist of little more than AGO1 and its loaded sRNA.…”

“…10,11 In addition, HEN1 and AGO1 also show some localization to the nucleus and to D-bodies. 11 In plants, the Slicer activity of AGO1 specifically co-elutes with a small protein complex, 19 to suggest that miRNA-loaded RISC may consist of little more than AGO1 and its loaded sRNA. Our molecular analyses strongly suggest that miRNA precursor transcripts, namely the primiRNA are the target of amiRNA-directed anti-miRNA silencing technology.…”

“…8,9 In specialized nuclear bodies, termed nuclear dicing or D-bodies, the initial miRNA precursor transcript processing event is catalyzed by DCL1 with the assistance of two other dsRNA-interacting proteins, SERRATE (SE) and dsRNA-BINDING DOMAIN1 (DRB1). [10][11][12][13] This smaller dsRNA molecule, the precursor-miRNA (pre-miRNA) transcript, undergoes a second cleavage step to release the miRNA/ miRNA* duplex from the stem-loop region. The second processing step of the Arabidopsis miRNA biogenesis pathway is also catalyzed by DCL1 in nuclear-localized D-bodies, however, DCL1 only requires the assistance of DRB1 to accurately direct this dicing event.…”

Reconstituting plant miRNA biogenesis

“…Many, if not all, of the pleitropic phenotypes of these mutants can be explained by miRNA biogenesis defects. Recently, several groups discovered that these three proteins can interact with each other in vitro and in vivo, and at least part of them colocalize in the same subnuclear bodies that also contain pri-miRNAs (15)(16)(17). The exciting work of Dong et al (13) nicely explains why HYL1 and SE are required for miRNA biogenesis.…”

The crosstalk between plant microRNA biogenesis factors and the spliceosome

“…43 Pri-miRNA polyadenylation site selection depends on the functional 5′ splice site Studies on plant pri-miRNA structures revealed that multiple polyadenylation sites might co-exist. 40,49,50 In the case of Arabidopsis pri-miR163, 2 polyA sites were identified: the proximal site that is located within the intron, and the distal site which is located at the end of the gene. 42,43 In the wild type A. thaliana cells, 60% of pri-miR163 is terminated at the distal site.…”