Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells

Abstract

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

Figure 1. Nuclear localization of human DICER1 protein.

(A) Western blot analysis for either cytoplasmic (Cyt) or nuclear (Nuc) extracts from 293T and HeLa cells using anti-DICER1, anti-LaminA and anti-GAPDH antibodies. LaminA and GAPDH were used as a nuclear or cytoplasmic marker protein, respectively. Each lane was loaded 50 µg of cytoplasmic extract or 100 µg of nuclear extract, respectively. (B) Western blot analysis for isolated nucleus with (+) or without (−) protease K treatment using anti-NUP214, anti-NUP153, anti-RNA polymerase II, anti-LaminA and anti-DICER1 antibodies. The signal intensity of each band was quantified using ImageJ software and intensity ratios were calculated from the “+” sample relative to the “−” sample. “Input” means the sample on 5% of volume used for immunoprecipitation (IP). (C) Confocal immunofluorescence images of DICER1 protein in HeLa cells without or with digitonin treatment. The signals of DICER1 protein (red) were detected using anti-DICER1 (12B5/4C6) antibody. Nuclei were counterstained with DAPI (blue) and cytoplasmic regions were co-stained with phalloidin (green). Scale bar represents 10 µm.

Figure 2. Co-immunoprecipitation (co-IP) of known DICER1-associated proteins with DICER1 protein in HeLa cells.

Co-IP experiments using anti-DICER1 (12B5/4C6) antibody from HeLa total cell extracts followed by Western blot analysis with indicated antibodies. “Input” means the sample on 5% of volume used for IP.

Figure 3. Co-IP of nuclear import receptor proteins with NUP153 protein.

(A) Co-IP experiments with NUP153 protein from cytoplasmic extracts of HeLa cells followed by Western blot analysis with indicated antibodies. “Input” means the sample on 5% of volume used for IP and “FT” indicates the samples on 5% of flow-through solution of IP samples. The asterisk shows the non-specific band using anti-GAPDH antibody. (B) Co-IP with NUP153 protein from nuclear extracts of HeLa cells followed by Western blot analysis with indicated antibodies.

Figure 4. Association of NUP153 protein with DICER1 protein in HeLa cells.

(A) Co-IP experiments from total cell extracts of HeLa cells followed by Western blot analysis with indicated antibodies. Endogenous NUP153 proteins were immunoprecipitated using anti-DICER1 antibody but not using mouse normal IgG (control). “Input” means the sample on 5% of volume used for IP. (B) In situ protein-protein associations between DICER1 and NUP153 were detected by Proximity Ligation Assay (PLA). HeLa cells were stained with mouse monoclonal anti-DICER1 and rabbit polyclonal anti-NUP153 antibodies and performed PLA. The association signals were detected by Duolink 100 Detection Kit 613 (red), and nuclei were counterstained with DAPI (blue). Samples co-stained with phalloidin (green) allow visualization of cell borders. Each red dot represents the detection of protein-protein association complex. White arrows indicate the signals at the nuclear periphery. Scale bar represents 10 µm. (C) PLA image shows the protein-protein associations between NUP153 and LaminA inside of nuclear membrane. (D) A negative control experiment of PLA was performed without addition of any primary antibodies.

Figure 5. Effects of siRNA knockdown against NUP153.

(A) Western blot analysis of negative control (NC) and NUP153 knockdown (KD) samples with indicated antibodies. Each lane was loaded 40 µg of cytoplasmic or nuclear extract, respectively. (B) Intensity ratio (Nuc/Cyt) of DICER1 protein in NUP153 KD sample is normalized to the intensity ratio of NC sample. The signal intensity of each band was quantified using ImageJ software. These plots show average values of the relative intensity ratio bracketed by s.e.m. error bars; calculated from three independent experiments. (C) Confocal immunofluorescence images in human fibroblasts transfected with NC or NUP153 siRNAs. The signals of NUP153 and DICER1 proteins were detected using rabbit polyclonal anti-NUP153 and mouse monoclonal anti-DICER1 antibodies, respectively. Nuclei were counterstained with DAPI. In merged figure, red, green and blue colors represent the signals of NUP153, DICER1 proteins and DAPI, respectively. Scale bar represents 10 µm.