Expression of TNRC6 (GW182) Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes

Abstract

The trinucleotide repeat containing 6 (TNRC6) family of proteins are core components of RNA interference (RNAi) and consist of three paralogs (TNRC6A, TNRC6B, and TNRC6C). The TNRC6 paralogs associate with argonaute (AGO) protein, the core RNAi factor, and bridge its interactions with other proteins. We obtained TNRC6A and TNRC6B single and double knockout cell lines to investigate how the TNRC6 paralogs contribute to RNAi. We found that TNRC6 proteins are not required for gene silencing when duplex RNAs are fully complementary. TNRC6 expression was necessary for regulation by a microRNA. TNRC6A, but not TNRC6B, expression was necessary for transcriptional activation by a duplex RNA targeting a gene promoter. By contrast, AGO2 is required for all three gene expression pathways. TNRC6A can affect the Dicer localization in cytoplasm versus the nucleus, but none of the three TNRC6 paralogs was necessary for nuclear localization of AGO2. Our data suggest that the roles of the TNRC6 paralogs differ in some details and that TNRC6 is not required for clinical therapeutic silencing mechanisms that involve fully complementary duplex RNAs.

Impact of scaffolding protein TNRC6 paralogs on gene expression and splicing

“…Double knockout TNRC6 AB-/-cells, had a significant increase in G2 phase in the TNRC6 AB-/-cells ( Figure 1B). The change in cell cycle stage for the double knock out cells is consistent with the reduced cell growth seen in the TNRC6 AB-/-cells (Liu et al, 2019).…”

“…TNRC6 A-/-or TNRC6 B-/-single knockout cells grew slower than wild cells (Liu et al 2019). TNRC6 AB-/-double knockout cells were the slowest proliferating.…”

“…We have previously described TNRC6 A-/-, TNRC6 B-/-, and TNRC6 AB-/-knockout cell lines (Liu et al 2019). HCT116 colorectal cancer cells were chosen as a parental line because they are diploid, which facilitates knocking out multiple genes simultaneously.…”

“…The knockout of the TNRC6A was confirmed by western blot analysis (Supplementary Figure 1A). We did not possess an adequate antibody to detect TNRC6B and knockout of TNRCB protein expression was confirmed by mass spectrometry (Liu et al 2019). The knockout of TNRC6 A was due to a point mutation that produced a frameshift while the knockout of TNRC6 B was achieved by a 95,481 base-pair deletion ( Figure 1A).…”

“…The TNRC6 paralogs are important binding partners for AGO proteins (Kalantari et al 2016b;Hicks et al 2017). The scaffolding domains of TNRC6 facilitate the recruitment of effector proteins for mRNA degradation (Piao et al 2010;Chekulaeva et al 2011;Fabian et al 2011;Hicks et al 2017) or transcriptional activation (Hicks et al 2017;Liu et al 2018;Liu et al 2019). Because of the partnership between AGO and TNRC6 we hypothesized that many gene expression changes would be shared between TNRC6…”