RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii
Sunday, 2016/09/25 | 06:16:16
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Tomohito Yamasaki, Masayuki Onishi, Eun-Jeong Kim, Heriberto Cerutti, and Takeshi Ohama SignificanceMicroRNAs are important regulators of gene expression in unicellular and multicellular eukaryotes. They are generally embedded in stem–loops of precursor transcripts and are excised by the dsRNA-specific nuclease DICER with the assistance of dsRNA-binding proteins. In animals and plants, proteins harboring two or three dsRNA-binding domains (dsRBDs) are involved in microRNA (miRNA) biogenesis. In contrast, we found that the Dull slicer-16 (DUS16) protein, which contains a single dsRBD and also an ssRNA-binding domain, is involved in miRNA biogenesis in the unicellular green alga Chlamydomonas. This finding sheds light on a molecular mechanism of miRNA biogenesis in unicellular organisms that may be similar to that in a common ancestor of animals and plants. AbstractCanonical microRNAs (miRNAs) are embedded in duplexed stem–loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.
See: http://www.pnas.org/content/113/38/10720.abstract.html?etoc PNAS September 20 2016; vol.113; no.38: 10720–10725
Fig. 1. Characterization of the DUS16 mutant. CC-124, wild-type strain; Gluc(1×), CC-124 transgenic strain expressing Gaussia luciferase (gluc) bearing a sequence perfectly complementary to Chlamydomonas miR9897-3p; dus16-1, DUS16-defective mutant of Gluc(1×); dus16-1(DUS16)-1 and -2, strains expressing HA-FLAG-tagged DUS16 in a dus16-1 background. (A) Schematic diagram of the gene structure and transcript of DUS16 (Cre05.g232004). dsRBD, dsRNA-binding domain; RRM, ssRNA-binding domain. (B–E) Each panel is representative of three independent experiments. (B) RT-PCR analysis in the indicated strains. Cre17.g697550 is a target of miR B-mediated cleavage. Cre16.g683650 is a target of miR C-mediated translation repression. A housekeeping gene, CBLP, was used as a control. (C) Northern blotting of gluc mRNA and the pri-miR9897 transcript in the indicated strains. Detection of CBLP was performed to check for equivalent loading of the RNA samples. (D) Immunoblotting of gluc, tagged DUS16, and AGO2/3 proteins in the indicated strains. The tagged DUS16 protein was detected with a monoclonal antibody against the HA tag. Both AGO2 and AGO3 polypeptides are recognized by a polyclonal antibody. Detection of histone H3 was performed to confirm similar loading of the protein samples. (E) Northern blotting of Chlamydomonas mature miRNAs in the indicated strains. U6 small nuclear RNA was used as a control for equal loading of the RNA samples. (F) Small RNA-seq analysis of CC-124 (green), Gluc(1×) (blue), and dus16-1 (red). Biological duplicates of sRNA libraries were generated from the indicated strains. sRNA reads, 17–25 nt in length, were classified according to their size. Absolute read counts, without normalization, are indicated. |
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