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Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Fredrik Söderbom)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Fredrik Söderbom)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2018 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 7, p. 937-954Article in journal (Refereed) Published
Abstract [en]

Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3’-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.

Place, publisher, year, edition, pages
UK: Taylor & Francis Group, 2018. Vol. 15, no 7, p. 937-954
Keywords [en]
Dicer, microRNA, amoeba, biogenesis, evolution, transcriptional start site, intron, development, Dictyostelium discoideum
National Category
Microbiology
Research subject
Biology
Identifiers
URN: urn:nbn:se:uu:diva-354016DOI: 10.1080/15476286.2018.1481697ISI: 000445654400013OAI: oai:DiVA.org:uu-354016DiVA, id: diva2:1220992
Funder
Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 621-2013-4665Carl Tryggers foundation , CST12:485Available from: 2018-06-19 Created: 2018-06-19 Last updated: 2018-10-18Bibliographically approved
In thesis
1. A small amoeba at the crossroads of the big RNAi world: MicroRNA biogenesis and Argonaute function in Dictyostelium discoideum
Open this publication in new window or tab >>A small amoeba at the crossroads of the big RNAi world: MicroRNA biogenesis and Argonaute function in Dictyostelium discoideum
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small non-coding RNA (ncRNA) mediated gene silencing, known as RNAi, is a key regulatory mechanism of gene expression in eukaryotes. MicroRNAs (miRNA), one major type of small ncRNAs, are about 21nt long and bound by Argonaute proteins. This RNA-protein complex, called RISC, silences post-transcriptionally target mRNAs containing partial or full complementary sequence to the miRNA.  

MiRNAs are generated from step-wise endonucleolytic cleavages of long primary transcripts (pri-miRNAs) by RNase III nucleases. Biogenesis of miRNAs differs between uni- and multicellular eukaryotes, and also between plants and animals. In this thesis, I aimed to understand miRNA maturation in the social amoeba Dictyostelium discoideum, which stands at the crossroads between these phylogenetically distant groups. We showed that Dicer-like protein DrnB is essential for global maturation of D. discoideum miRNAs. The study of two pri-miRNAs revealed the conserved 5’ m7G-cap structures, but different 3’end formation from each other, and also from canonical miRNAs in plants and animals. In agreement with its evolutionary position, D. discoideum miRNA biogenesis showed unique and also shared features with both life groups.

D. discoideum grows as a unicellular organism, but can switch to a multicellular development upon starvation. Most miRNAs, many other small ncRNAs, and Argonaute proteins, the core effectors of the RISC, are differentially expressed during development, indicative of a crucial role of RNAi mediated regulation throughout D. discoideum life cycle. Among the five Argonaute homologs in D. discoideum, I investigated the functions of three members, e.g. AgnB, C and E. Judging from their subcellular localization, the phenotypic consequences and transcriptional alteration resulting from single Argonaute gene deletion, our results suggested different roles of AgnB, C and E. Possibly AgnB associates with miRNAs and regulates gene expression post-transcriptionally; while AgnC seems to be involved in nuclear RNAi. Finally, the cytoplasmic AgnE inhibits D. discoideum cell growth and regulates developmental timing via an unknown mechanism.

My thesis work expands our knowledge on D. discoideum RNAi with focuses on miRNA biogenesis and potential function of Argonaute proteins and, all together, sheds lights on the evolution of miRNA and RNAi.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 73
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1686
Keywords
RNAi, microRNA, Argonaute protein, miRNA biogenesis, Dictyostelium discoideum, social amoeba, Dicer, development, growth
National Category
Microbiology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-354334 (URN)978-91-513-0371-0 (ISBN)
Public defence
2018-09-12, B22, Uppsala Biomedicinska Centrum, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-08-16 Created: 2018-06-19 Last updated: 2018-08-27

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Liao, ZhenHöppner, Marc P.Grabherr, ManfredSöderbom, Fredrik

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