RNAi Factors Are Present and Active in Human Cell Nuclei

Keith T Gagnon,Liande Li,Yongjun Chu,Bethany A Janowski,David R Corey

doi:10.1016/j.celrep.2013.12.013

Keith T Gagnon, Liande Li + Show 3 more

Open Access

https://doi.org/10.1016/j.celrep.2013.12.013

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Abstract

SummaryRNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus.

Highlights

Since the discovery of mammalian RNA interference (RNAi) (Elbashir et al, 2001), over 50,000 reports have described the use of small interfering RNAs
We began our study by using HeLa cells to examine the localization of Ago2, the catalytic core of RNAi (Liu et al, 2004)
We used wide-field immunofluorescence microscopy to test localization of Dicer, TAR RNA-binding protein (TRBP) and TNRC6A and observed staining in the nucleus as well as in the cytoplasm (Figure S1I-K). These results using different microscopy platforms, cell lines, and detection reagents suggest the nuclear presence of the protein machinery that enables RNAi

Summary

Introduction

Since the discovery of mammalian RNA interference (RNAi) (Elbashir et al, 2001), over 50,000 reports have described the use of small interfering RNAs (siRNAs). Almost all of these studies have assumed that the regulation of RNAi and its silencing activity occurs in the cytoplasm (Gurtan and Sharp, 2013). What role the nuclear compartment might play in the regulation of RNAi pathways is unknown. These uncertainties have significantly hampered investigation of nuclear RNA biology and the development of nuclear RNAi as a laboratory tool and potential therapeutic. Nuclear RNAi activity and localization of RNAi factors to the nucleus have been reported previously, questions about the purity of cell extracts (Holding, 2005), the resolution of localization studies, and nucleocytoplasmic transport of the RNA targets and products of RNAi have kept nuclear RNAi a controversial subject

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