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

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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.