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. 2016 Jan 29;291(5):2302-9.
doi: 10.1074/jbc.C115.695049. Epub 2015 Dec 23.

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

Nishi R Sharma  1 , Xiaohong Wang  1 , Vladimir Majerciak  1 , Masahiko Ajiro  1 , Michael Kruhlak  2 , Craig Meyers  3 , Zhi-Ming Zheng  4
Affiliations

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

Nishi R Sharma et al. J Biol Chem. .

Abstract

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.

Keywords: Ago2; RNA-binding protein; cervix; keratinocyte; keratinocytes; microRNA (miRNA); microscopic imaging; nuclear distribution; papillomavirus; virus.

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Figures

FIGURE 1.
FIGURE 1.
Partial nuclear distribution of Ago2 in HeLa and HaCaT cells. A, specificity of anti-Ago2 antibody in Western blotting of Ago2 expressed from HeLa cells transfected with a nonspecific (−) or Ago2-specific (+ si-Ago2) siRNA. Total cell extract (5 μl) was used for the assay, and β-actin served as a loading control. Numbers on the left are protein molecular mass markers in kDa. B and C, subcellular distribution of Ago2 in HeLa (B) and HaCaT (C) cells. Immunofluorescence staining of endogenous Ago2 (green) was performed with an anti-Ago2-specific antibody tested by Western blotting (A). Nuclei were counterstained with Hoechst dye (blue). D, stress-induced translocation of cytoplasmic Ago2 into TIA-1-positive stress granules. E, colocalization of Ago2 with TIA-1 in stress granules upon arsenite treatment. HeLa cells with or without arsenite treatment were simultaneously stained with anti-Ago2 (green) and anti-TIA-1 (red) antibodies. The nuclei were stained with Hoechst stain. All images were captured by confocal microscopy. Scale bar = 10 μm. Dashed boxes represent selected zoomed area.
FIGURE 2.
FIGURE 2.
Nuclear distribution of Ago2 in HFK and their derived organotypic (raft) cultures. A and B, subcellular Ago2 distribution in untransfected (A) or siRNA (nonspecific (si-NS) or Ago2-specific (si-Ago2)) -transfected primary HFKs (B). Endogenous Ago2 was detected by immunostaining with an anti-Ago2 antibody (green). Nuclei were counterstained with Hoechst dye for DNA. Images were captured by confocal microscopy. Scale bar = 10 μm in A and 20 μm in B. C, subcellular ago2 was examined in nuclear (N) and cytoplasmic (C) fractions of HeLa and primary HFKs by Western blotting using a mouse anti-Ago2 antibody. Cytoplasmic tubulin and nuclear hnRNP C1/C2 were used to provide fractionation efficiency. The relative amounts of Ago2 in the nuclear fraction (being set to 1) over the cytoplasmic fraction are shown at the bottom of the Ago2 blot after normalizing with nuclear hnRNP C1/C2 and cytoplasmic tubulin, respectively. D, purified HA-Ago2 protein for antibody absorption assays. Recombinant HA-Ago2 protein was purified from the cellular extract of HEK293 cells by an anti-HA antibody affinity purification. The purity of HA-Ago2 (arrow) in elution fractions (E1–3) was determined by SDS-PAGE with silver staining. E and F, subcellular Ago2 distribution in HFK-derived raft cultures without (E, HFK) or with HPV16 (F, HFK16) infection. Endogenous Ago2 was detected by immunostaining with an anti-Ago2 antibody (green). Ago2-absorbed anti-Ago2 antibody in F and secondary antibody only (2o Ab only, F) served as a negative control staining. Nuclei were counterstained with Hoechst dye. DIC, differential interference contrast image. Images were captured by confocal microscopy. Scale bar = 20 μm in E and F. Dashed boxes represent zoomed areas. G, quantification of the cells with nuclear Ago2 staining in HFK raft cultures without or with HPV16 (HFK16) or HPV18 (HFK18) infection. Mean + S.D. from 150 cells in each condition from three independent experiments. 1o, mouse anti-Ago2 antibody; 2o, rabbit anti-mouse IgG antibody.
FIGURE 3.
FIGURE 3.
Nuclear distribution of Ago2 in HFK raft tissues defined by lamin A/C staining. A, immunostaining for endogenous Ago2 and lamin A/C in HFK raft tissues without (HFK) or with HPV16 (HFK16) infection by anti-Ago2 (green) and anti-lamin A/C (red) antibodies. Nuclei were counterstained with Hoechst dye for DNA. Images were captured by confocal microscopy. Scale bars = 20 μm. B, orthogonal view (indicated by a vertical or horizontal line) of a z-stack of HPV16-infected HFK raft tissues showing the subcellular distribution of Ago2 (green), lamin A/C (red), and DNA (blue) in double immunostaining in A. C, the distribution of signal intensities for Ago2, lamin A/C, and DNA over three selected cells from panel B (arrows) using the ZEN 2 imaging software. AU, arbitrary units.
FIGURE 4.
FIGURE 4.
Tissue-context nuclear distribution of Ago2. A, immunostaining of endogenous Ago2 in normal cervix, skin, and larynx by anti-Ago2 antibody. B and C, distinguishable subcellular distribution of endogenous Ago2 and PABPC1. Normal or cancerous cervical tissues were double-stained with anti-Ago2 (green) and anti-PABPC1 (red) antibodies (B). Nuclei were counterstained with Hoechst dye (A and B). All images were captured by confocal microscopy. Scale bars = 20 μm. Dashed boxes represent zoomed areas. The levels of Ago2 and PABPC1 in the selected cells from normal cervix were measured by a line crossing over the stained cells. Nuclear DNA counterstained with Hoechst dye served to define the cell nuclear and cytoplasmic border (C). The intensity plot of individual measurements in arbitrary units (AU) was shown after normalization to maximum intensities.
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