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Systemic RNAi Relies on the Endomembrane System in Caenorhabditis elegans
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Andrea Hinas)ORCID iD: 0000-0001-8905-7561
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

The membrane system of a eukaryotic cell is a large and complex system handling the transport, exchange and degradation of many kinds of material. Recent research shows that double-stranded RNA (dsRNA) mediated gene silencing (RNA interference) is a membrane related process. After long dsRNA is processed to small interfering RNA (siRNA) by Dicer, the guide strand and passenger strand are separated in the RNA induced silencing complex (RISC) by Argonaute. The process of loading siRNA into RISC has been suggested to occur at the rough Endoplasmic Reticulum (rER).The components of RISC also associate with late endosomes/multivesicular bodies (MVBs). Furthermore, disturbing the balance between late endosomes/MVBs and lysosomes has been shown to affect the efficiency of silencing.

We use the nematode Caenorhabditis elegans as our model organism to study two questions: how does membrane transport affect RNAi and spreading of RNAi from the recipient cells to other tissues (systemic RNAi); and how does RNA transport contribute to the multigenerational silencing induced by dsRNA (RNAi inheritance)? Using SID-5, a protein required for efficient systemic RNAi, as bait in a yeast two-hybrid (Y2H) screen, we got 32 SID-5 interacting candidate proteins. Two of these are the SNARE protein SEC-22 and the putative RNA binding protein C12D8.1. In two additional Y2H screens, we found that SID-5 interacts with multiple syntaxin SNAREs, including SYX-6, whereas SEC-22 only interacts with SYX-6. SNAREs usually function in vesicle fusion processes. We found the two SNARE proteins SEC-22 and SYX-6 to be negative regulators of RNAi and to localize to late endosomes/MVBs. In addition, loss of sid-5 leads to an endosome maturation defect. Finally, we found that the putative RNA binding protein C12D8.1 negatively regulates RNAi inheritance and that C12D8.1 mutant animals show impaired RNAi upon targeting a new gene. Taken together, the results presented in this thesis provide us with more evidence for the connection of the membrane transport system and RNAi. The identification of a putative negative regulator of RNAi inheritance further enriches this research field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1571
Keyword [en]
Systemic RNAi, RNAi inheritance, late endosomes/MVBs, SID-5, SEC-22, SYX-6, C12D8.1
National Category
Microbiology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-330894ISBN: 978-91-513-0093-1 (print)OAI: oai:DiVA.org:uu-330894DiVA, id: diva2:1147486
Public defence
2017-11-23, B42, Uppsala Biomedical central, Husagatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2017-11-01 Created: 2017-10-06 Last updated: 2017-11-01
List of papers
1. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans
Open this publication in new window or tab >>The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans
2017 (English)In: RNA, ISSN 1355-8382, Vol. 23, no 3, p. 297-307Article in journal (Refereed) Published
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-321388 (URN)10.1261/rna.058438.116 (DOI)
Available from: 2017-05-04 Created: 2017-05-04 Last updated: 2017-10-13
2. RNA Transport Protein SID-5 Interacts with Multiple SNAREs and Affects Membrane Trafficking in C. elegans Intestinal Cells
Open this publication in new window or tab >>RNA Transport Protein SID-5 Interacts with Multiple SNAREs and Affects Membrane Trafficking in C. elegans Intestinal Cells
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-320894 (URN)
Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2017-10-13
3. Putative RNA-Binding Protein C12D8.1 Negatively Regulates Inheritance of RNAi in Caenorhabditis elegans
Open this publication in new window or tab >>Putative RNA-Binding Protein C12D8.1 Negatively Regulates Inheritance of RNAi in Caenorhabditis elegans
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-320895 (URN)
Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2017-10-13

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