A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells
Castanotto, Daniela, Lin, Min, Kowolik, Claudia, Wang, LiAnn, Ren, Xiao-Qin, Soifer, Harris, Koch, Troels, Hansen, Bo Rode, Armstrong, Brian, Wang, Zhigang, Bauer, Paul, Rossi, John and Stein, CA (2015) A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells. Science Advances, 43 (19). pp. 9350-9361. ISSN 1362-4962
Abstract
A longstanding belief, generated from the use of transfection reagents to internalize oligonucleotides (ASOs) in cells, is that ASOs exclusively trigger mRNA degradation in the nucleus. In contrast, we have identified a novel cytoplasmic mechanism through which ASOs silence their targets when delivered gymnotically (i.e., in the absence of any transfection reagent). Our data support the existence of a productive, PKC--dependent endocytotic pathway which leads to the interaction of the ASO with the Ago-2 PAZ domain followed by localization of the ASO-Ago2 complex into GW-182 mRNA degradation cytoplasmic bodies (GW-bodies). The degradation products of the targeted mRNA are not generated by Ago-2-directed cleavage and do not seem to exhibit a typical RNase H endonucleolytic cleavage pattern, indicating that other nucleases may be involved. Blocking the evolution of late endosomes into multi-vesicular bodies reduces ASO access to Ago-2 and results in potent reduction of ASO silencing ability. The identification of a cytoplasmic pathway occurring after gymnotic ASO delivery complements the previously known nuclear localization of ASO activity after transfection.
Item Type: | Article |
---|---|
Date Deposited: | 26 Apr 2016 23:45 |
Last Modified: | 26 Apr 2016 23:45 |
URI: | https://oak.novartis.com/id/eprint/24308 |