uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
The Dictyostelium discoideum Argonaute protein AgnE regulates cell growth
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, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Argonaute proteins play essential roles in the RNA interference (RNAi) pathways in eukaryotes. The members of this conserved class of proteins are guided to their target RNAs by their associated small RNAs and can thereby regulate gene expression. The number and function of Argonautes varies depending on the organism but it is clear that they together with their interacting small RNAs constitute the core of the RNA induced silencing complex, RISC. Little is known about the Argonautes in the social amoeba Dictyostelium discoideum, a unicellular organism that can go through multicellular development and evolutionary is placed between plants and animals. In this study, we investigated the phenotypic consequences of deleting the genes for three Argonautes, AgnB, AgnC, and AgnE in D. discoideum. All three Argonautes have an effect on growth since depletion of AgnB and AgnC impaired growth while AgnE depletion, surprisingly, resulted in faster cell division. The intriguing role of AgnE in growth regulation prompted us to further study this protein. We expressed an AgnE-GFP fusion protein and showed that this localize in the cytoplasm. High-throughput sequencing of mRNA from growing agnE- and wt cells showed that genes required for the nucleobase biosynthetic process as well as genes for ribosomal proteins are upregulated in the agnE knock-out strain, which is in line with its faster growth rate. Furthermore, the RNAi related gene encoding RNA-dependent RNA polymerase C is downregulated, which may result in accumulation of miRNAs. The possible connection between growth rate and miRNA levels was explored by analyzing growth rate in a strain depleted of miRNAs, i.e. where the gene for the Dicer-like protein DrnB had been knocked out. This strain grew much slower than wt and this phenotype could not be rescued by disrupting agnE in the drnB- background. This suggests that DrnB acts upstream of AgnE in regulating D. discoideum growth.

Keywords [en]
RNAi, Argonautes, microRNAs, small RNAs, mRNA-seq
National Category
Microbiology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-354019OAI: oai:DiVA.org:uu-354019DiVA, id: diva2:1220997
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: 2018-06-19
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

Open Access in DiVA

No full text in DiVA

Authority records BETA

Söderbom, Fredrik

Search in DiVA

By author/editor
Söderbom, Fredrik
By organisation
MicrobiologyScience for Life Laboratory, SciLifeLab
Microbiology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 229 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf