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.

Keywords: RNA interference; argonaute; nuclear/cytoplasm localization; trinucleotide repeat containing 6.

FIG. 1.

Diagrams of TNRC6 protein paralogs' domains and mutations. (A) The major isoforms of TNRC6A (isoforms 1, 2, 5, and 6) have been mutated by insertion of 1 base pair into the AGO binding domain region. (B) The major isoforms of TNRC6B have a large 95,481 base-pair deletion of the AGO binding domain region. TNRC6B isoform 3 does not contain the AGO binding region and is not affected by this deletion. (C) Two isoforms of TNRC6C. AGO, argonaute; TNRC6, trinucleotide repeat containing 6.

FIG. 2.

Diagram of the small RNA systems used to evaluate TNRC6 involvement in RNAi processes. (A) AGO2 loaded with siATX-3 targets and cleaves ATX-3 mRNA causing siRNA knockdown of ATX-3 in the cytoplasm. (B) AGO2 loaded with siMalat1 targets and cleaves Malat1 ncRNA causing siRNA knockdown of Malat1 in the nucleus. (C) AGO2 loaded with a small RNA binds to a sense transcript that overlaps the COX-2 promoter. This causes further activation of gene transcription. (D) AGO2 loaded with miR34a targets and causes the degradation of Sirt1 mRNA. This causes the activation of P53 and apoptosis. ATX-3, ataxin-3; COX-2, cyclooxygenase-2; mRNA, messenger RNA; miRNA, microRNA; ncRNA, noncoding RNA; RNAi, RNA interference; siRNA, small interfering RNA.

FIG. 3.

Protein and gene expression level of TNRC6 and AGO paralogs. (A) Table of protein copies for TNRC6 paralogs, AGO paralogs, and housekeeping genes. (B) mRNA levels for TNRC6 paralogs, n = 3. (C) mRNA level of AGO paralogs, n = 2. Error bars represent standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001. NC, no confidence; ND, not detected; WT, wild-type. Data in graph is in order as follows: WT, Ago2^−/−, TNRC6 A^−/−, TNRC6 B^−/−, TNRC6 AB^−/−.

FIG. 4.

AGO2 and TNRC6 protein cellular distribution in different cell lines. (A). Western blot of cytoplasmic and nuclear fractions from WT, AGO2^−/− and TNRC6A^−/− of calnexin, an ER protein, tubulin, a cytoplasm marker, and histone H3, a chromatin protein to show the purity of the extraction. (B) Western blot of cytoplasmic and nuclear fractions from WT, TNRC6B^−/−, and TNRC6AB^−/− showing purity of the extraction. ER, endoplasmic reticulum.

FIG. 5.

Assay of siRNA pathways in TNRC6 knockout cell lines. (A) siRNA-mediated inhibition of nuclear noncoding RNA MALAT1 expression, n = 3. Each measurement using siMALAT1 is normalized to treatment with the corresponding siCM control RNA. (B) Western blot analysis of siRNA-mediated inhibition of ATX3 protein expression. Error bars represent standard deviation, ***P < 0.001. siCM and siGL2 are noncomplementary duplex RNAs.

FIG. 6.

Assay of miRNA pathway in TNRC6 knockout cell lines. (A) Schematic of miR34a pathway. (B) Quantification of live cells after treating cells with an miR34, n = 3. Each treatment with the miR-34a mimic is normalized to a parallel treatment with a non complementary RNA duplex. Error bars represent standard deviation, *P < 0.05; **P < 0.01; ***P < 0.001. siGL2 is a non complementary control duplex RNA.

FIG. 7.

Effect of knocking out TNRC6 paralogs on transcriptional activation of COX-2 protein expression by a promoter-targeted duplex RNA. (A) Western blot analysis of activation of COX-2 protein expression, n = 2. (B) Quantification of data in (A). Error bars represent standard deviation, *P < 0.05; **P < 0.01. siGL2 is a non complementary control duplex RNA.