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Connecting Systemic RNAi to the Endomembrane System in Caenorhabditis elegans
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Hinas)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

RNA interference (RNAi) is a gene regulation mechanism conserved among eukaryotes. To silence gene expression, RNAi relies on a short single-stranded guide RNA to steer the RNA-induced Silencing Complex (RISC) to mRNAs with guide strand-complementary sequences. RNAi is a highly membrane-associated process. The RISC complex is likely loaded at the rough Endoplasmic Reticulum, where it can bind to and degrade mRNAs. Components of the RISC complex also colocalize to late endosomes, and the efficiency of RNAi-mediated silencing is affected by changes in late endosome to lysosome fusion. RNAi can be systemic and inherited, effecting gene silencing in distal tissues and in the offspring.

In this thesis, the model organism Caenorhabditis elegans was used to identify and characterize factors connecting systemic and inherited RNAi to the endomembrane system. We identify two SNARE proteins, SEC-22 and SYX-6, that both act as negative regulators of RNAi. SNAREs are necessary for vesicle fusion. Both SEC-22 and SYX-6 localize to late endosomes, and both interact with systemic RNAi protein SID-5 in a yeast two-hybrid (Y2H) screen. We find that in addition to its function in systemic RNAi, SID-5 is required for proper maturation of late endosomes. Furthermore, we identify the putative RNA-binding protein C12D8.1 as a novel regulator of RNAi inheritance. Mutant C12D8.1 animals will have enhanced inheritance of RNAi silencing, which negatively affects the ability of the progeny to silence new targets using RNAi. Finally, we describe a novel, object-based method for estimating significance in colocalization studies. This method helped us describe and quantify spatial relations between fluorophore-labeled proteins in situations where such analyses would otherwise be impossible.

In conclusion, the work presented here further elucidates the connection between cellular RNAi, the endomembrane system, and the outside world.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1526
Keywords [en]
Systemic RNAi, RNAi inheritance, Endomembrane system, Late endosome, SID-5, SEC-22, SYX-6, C12D8.1, RNA
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-320897ISBN: 978-91-554-9932-7 (print)OAI: oai:DiVA.org:uu-320897DiVA, id: diva2:1091550
Public defence
2017-06-12, A1:111a, Uppsala Biomedicinska Centrum, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2017-05-19 Created: 2017-04-27 Last updated: 2017-06-08
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
4. DESCROD Estimates Significant Colocalization by Randomized Object Displacement
Open this publication in new window or tab >>DESCROD Estimates Significant Colocalization by Randomized Object Displacement
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-320893 (URN)
Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2017-04-27

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