Human GW182 Paralogs Are the Central Organizers for RNA-Mediate Control of Transcription

RNA interference (RNAi) is an endogenous mechanism for regulating gene expression that can be manipulated for experimental or therapeutic purposes to knockdown protein expression. In the mammalian cell cytoplasm, small RNAs direct RNAi proteins to inhibit translation by regulating mRNA stability. In non-mammalian cell nuclei, RNAi proteins control gene transcription. Recently, in mammalian cell nuclei, the RNAi proteins were shown to control transcription and splicing. Despite what is known about RNAi factors in non-mammalian cell nuclei, the mechanisms of mammalian RNA-mediated nuclear regulation are not well understood and remain controversial, hindering the effective application of nuclear RNAi and blinding investigation of its natural regulatory roles. Argonaute 2 (AGO2) and TNRC6A (GW182) are the core proteins of RNAi. To better understand RNAi-mediated nuclear gene regulation, my goal was to use semi-quantitative (SINQ) mass spectrometry analyses on purified protein complexes to build a protein interaction network of nuclear AGO2 and TNRC6A. The stable interactions of AGO2 detected with this protocol were the TNRC6A, B, and C paralogs. While this did not reveal many proteins, it did provide a new direction to take with mass spectrometry analyses. Since the TNRC6 paralogs are stable interacting partners, analysis on those protein complexes were performed to reveal a new shell of nuclear RNAi interactions. Mass spectrometry of TNRC6A protein complexes revealed these proteins are central to forming interactions between the RNAi machinery and many proteins involved in transcriptional regulation. TNRC6A interactions include the AGO proteins, CCR4-NOT complex, histone modifiers, and the mediator complex. In addition, novel interactions with four DNA damage repair proteins were identified, providing another direction for future investigation. Functional analysis revealed that TNRC6, AGO2, NAT10 (histone acetylation), WDR5 (H3K4me3), and MED14 (Mediator) proteins are involved in RNA-mediated COX-2 transcriptional activation. Taken together, the mass spectrometry and functional experiments provide evidence that these RNA-AGO-TNRC6 complexes act globally in cell nuclei to regulate transcription. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. The significance of my data is that it can lead to new advances in RNAi capabilities beyond canonical applications.