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
Functional analyses of RNA interference effectors in Dictyostelium discoideum during growth and development
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]

RNA interference (RNAi) is a widespread biological process, which regulates gene expression in eukaryotic cells. A complex of proteins and small RNAs, RISC, mediates this gene regulation. Central to the function of RISC are the Argonaute effector proteins, which bind the small RNAs, e.g. micro (mi)RNAs and small interfering (si)RNAs. It has previously been shown that RNAi is important to control transposon mobilization in the social amoeba Dictyostelium discoideum, a unicellular eukaryote that upon starvation enters a multicellular developmental program. Information concerning the five Argonautes in D. discoideum is scarce but several of them appear to inhibit transposon mobilization by RNAi related mechanisms. In a recent study, we showed that three of the Argonautes in D. discoideum are involved in controlling cell division. In this study, we perform mRNA- and small RNA-seq. from growing and developing cells, combined with phenotypic studies of D. discoideum strains depleted of AgnB, AgnC, and AgnE. The previously observed effect on cell division, i.e. faster growth for agnE-, and slower for agnB- and agnC- cells, is associated with increased and decreased expression, respectively, of genes involved in nucleotide metabolism. Furthermore, all three argonautes appear to be involved in downregulation of ribosomal protein genes during development while AgnE also contributes to reduced expression of protein coding genes during growth. These effects are likely mediated by small RNAs. We further report the subcellular localization of the three Argonautes, where AgnB is mainly localized in the cytoplasm, AgnC in the nucleus, and the previously reported cytoplasmic localization for AgnE was confirmed. Finally, we present data indicating that AgnB is interacting with miRNAs, suggesting that this Argonaute is involved in miRNA mediated gene regulation in D. discoideum.  Taken together, our data indicate that none of the Argonautes components are essential for cell division and development, but all participate in fine-tuning of gene expression for optimal growth and synchronous multicellular development.

Keywords [en]
RNAi, microRNA, Argonautes, Dictyostelium discoideum, amoeba, development
National Category
Cell Biology Microbiology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-354021OAI: oai:DiVA.org:uu-354021DiVA, id: diva2:1221016
Funder
Swedish National Infrastructure for Computing (SNIC), 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
Cell BiologyMicrobiology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 166 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