Abstract
In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.
Highlights
MicroRNAs are found in mammalian nuclei, as are key RNAi proteins like AGO2 and the GW182 paralogs TNRC6A, TNRC6B, and TNRC6C (Gagnon et al, 2014; Matsui et al, 2015)
Immunoprecipitation and Mass Spectrometry for TNRC6A Nuclear extracts were prepared for large-scale immunoprecipitations, typically requiring several hundred million cells
After immunoprecipitation using anti-TNRC6A antibody and an anti-rabbit immunoglobulin G (IgG) antibody as a control, the purified samples were analyzed by western blot to confirm the presence of TNRC6A (Figure S1D)
Summary
MicroRNAs (miRNAs) are found in mammalian nuclei, as are key RNAi proteins like AGO2 and the GW182 paralogs TNRC6A, TNRC6B, and TNRC6C (Gagnon et al, 2014; Matsui et al, 2015). The presence of both small RNAs and RNAi factors in nuclei suggests that RNA-mediated recognition may regulate RNA-dependent processes like transcription or splicing. There have been reports that miRNAs and duplex RNAs can affect gene transcription (Weinberg and Morris, 2016; Kalantari et al, 2016a) While these reports have built a strong case for nuclear RNAi function, the detailed mechanism for transcriptional regulation has not been characterized, blocking progress toward understanding the broader significance of nuclear RNAi or resolving critical unanswered questions regarding the roles it might play in normal physiology and development. The narrow range of proteins that interact with AGO2 was insufficient to explain the observed functional control of small RNAs on transcription
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