Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading

Abstract

RNAi is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We include a method for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. This protocol facilitates the characterization of nuclear RNAi, and it can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4-6 d to complete.

Figure 1

Schematic of cell fractionation and extract preparation steps of this protocol.

Figure 2

Schematic of the in vitro Ago2 loading assay described in this protocol.

Figure 3

Quality assessment of subcellular fractionation. (A) Western blot analyses of cytoplasmic (Cyto) and nuclear (Nuc) fractions. Western analysis protocol followed is from Gagnon and colleagues. (B) Fluorescence microscopy of isolated nuclei. Blue is DAPI stain, which binds chromatin DNA. Yellow is ER Tracker Red dye pseudocolored yellow, which binds sulfonlyurea receptors on the ER membrane surface. Scale bar = 5 μm.

Figure 4

In vitro Ago2 loading assay and typical results from Step 9B. (A) Illustration of in vitro Ago2 loading assay procedure described in Step 9B. (B) Denaturing polyacrylamide gel electrophoresis of Ago2-bound small RNAs from typical cytoplasmic (cyto) and nuclear (nuc) loading reactions. (C) Native polyacrylamide gel electrophoresis of small RNAs that co-purified with IgG or Ago2 antibody. Markers used are single-stranded guide siRNA (s.s. guide) and duplex siRNA (duplex) loaded in separate lanes to the left.