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. 2013 Jan;19(1):17-35.
doi: 10.1261/rna.034769.112. Epub 2012 Nov 13.

Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus

Kenji Nishi  1 , Ai Nishi, Tatsuya Nagasawa, Kumiko Ui-Tei
Affiliations

Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus

Kenji Nishi et al. RNA. 2013 Jan.

Abstract

GW182 family proteins play important roles in microRNA (miRNA)-mediated gene silencing. They interact with Argonaute (Ago) proteins and localize in processing bodies, which are cytoplasmic foci involved in mRNA degradation and storage. Here, we demonstrated that human GW182 paralog, TNRC6A, is a nuclear-cytoplasmic shuttling protein, and its subcellular localization is conducted by a nuclear export signal (NES) and a nuclear localization signal (NLS) identified in this study. TNRC6A with mutations in its NES region was predominantly localized in the nucleus in an Ago-independent manner. However, it was found that TNRC6A could bring Ago protein into the nucleus via its Ago-interacting motif(s). Furthermore, miRNAs were also colocalized with nuclear TNRC6A-Ago and exhibited gene silencing activity. These results proposed the possibility that TNRC6A plays an important role in navigating Ago protein into the nucleus to lead miRNA-mediated gene silencing.

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Figures

FIGURE 1.
FIGURE 1.
Subcellular localization of TNRC6A fragments and identification of NES and NLS. (A) Schematic representation of TNRC6A deletion mutants, which were expressed as fusion proteins with myc-GFP. Numbers denote the amino acid position relative to the N terminus. GW-I, -II, and -III (black boxes) indicate GW-repeated Ago-binding motifs. NLS (blue box) and NES (red box) were identified in this study. B–I, K, and M at the right side indicate the fragments used in B–I, K, and M. (WT) Wild type. (B–N) Subcellular localization of the indicated myc-GFP-tagged TNRC6A fragments (B–M) and the control myc-GFP (N). Fluorescent microscopy images of GFP are shown in the left panels, and their merged images with DAPI are shown in the right panels, in which the GFP signal is shown in white and the DAPI signal in cyan. An arrow in F indicates the cell in which myc-GFP-TNRC6A-(925–1709) signals are exclusively observed in the nucleus. (*) Cells not transfected with each expression construct. Bars, 20 μm. (O,P) Sequences of the TNRC6A NLS motif and its mutant (NLS-mut) (O), and TNRC6A NES and its mutant (NES-mut) (P). The red letters represent basic amino acids or hydrophobic residues. The blue letters indicate substituted amino acids. (Q) The ratio of cells expressing GFP exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow). B–N at the upper side indicate the fragments used in B–N. A blue asterisk indicates that the expression of GFP is strongly observed in the nucleus, although a negligible signal is also observed in the cytoplasm.
FIGURE 2.
FIGURE 2.
Subcellular localization of NES and NLS mutants of TNRC6A in the full-length context. (A–D) Subcellular localization of the myc-GFP-TNRC6A (WT; A), myc-GFP-TNRC6A-NLS-mut (B), myc-GFP-TNRC6A-NES-mut (C), and myc-GFP-TNRC6A-NLS/NES-mut (D) proteins. Bars, 20 μm. (E) The ratio of the cells transfected with pmyc-GFP-TNRC6A and its mutants by classification according to their subcellular localization. Protein expression was verified by Western blot (Supplemental Fig. S1B). (F) The schematic diagram of observation of the cells expressing myc-GFP-TNRC6A-NES-mut by confocal microscopy. (G) Confocal images of the myc-GFP-TNRC6A-NES-mut at each section shown in F. Bar, 10 μm.
FIGURE 3.
FIGURE 3.
Nuclear export of TNRC6A by Exportin 1. (A–D) Fluorescent images of HeLa cells transfected with pmyc-GFP-TNRC6A (WT; A,C) or pmyc-GFP-TNRC6A-(935–984) (B,D), treated with LMB (A,B), or siXPO1 and siXPO5 (C,D). Bars, 20 μm. (E,F) The ratio of cells expressing GFP exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow). A and B (E), and C and D (F) at the upper side indicate the results shown in A and B, and C and D, respectively. (G,H) Western blot of Exportin 1 (G) or Exportin 5 (H) in HeLa cells transfected with siControl and siXPO1, or siControl and siXPO5, respectively. Anti-tubulin antibody was used as a loading control. (I) HeLa cells treated with and without LMB for 8 h were harvested, and cell fractionation was performed. Ten μg of the cytoplasmic fraction (C), 5 μg of the nuclear soluble fraction (NS), or 10% of the nuclear insoluble fraction (NI) were analyzed by Western blot using an anti-TNRC6A antibody. Blue and red arrowheads indicate the positions corresponding to ∼210-kDa TNRC6A (NP_055309) and ∼182-kDa TNRC6A (AAK62026) proteins, respectively. Anti-tubulin, anti-Lamin A/C, and anti-Histone H3 antibodies were used as a cytoplasmic marker, a nuclear soluble and insoluble marker, and a nuclear insoluble marker, respectively. Asterisks (*1, *2, *3) indicate nonspecific bands. (J) GST pulldown assay of Exportin 1 and TNRC6A fragments. Equal amounts of His-GST, His-GST-TNRC6A-(935–984), or His-GST-TNRC6A (935–984)-NES-mut were immobilized on glutathione-sepharose beads and incubated with recombinant Exportin 1 with or without Ran(Q69L)GTP. Bound fractions were analyzed by Western blot using an anti-XPO1 antibody. The lower panel is the pattern of Coomassie brilliant blue (CBB) staining, which indicates that each protein is similarly immobilized. (K) GST-TNRC6A-(935–984) was immobilized on glutathione-sepharose beads and incubated with the indicated amount of LMB treated-Exportin 1 in the presence of Ran(Q69L)GTP. Bound fractions were analyzed by Western blot using anti-XPO1. The lower panel shows the CBB staining pattern.
FIGURE 4.
FIGURE 4.
Nuclear transport of TNRC6A is Ago2-independent. (A) Schematic representation of myc-GFP-TNRC6A-NES-mut with GW motif deletions. Note that NES regions were mutated. (B) Fluorescent images of HeLa cells transfected with the expression constructs of myc-GFP-TNRC6A (WT) or myc-GFP-TNRC6A-NES-mut with GW motif deletions and stained by an anti-Ago2 antibody, followed by Cy5-conjugated anti-mouse IgG. Fluorescent images are shown, from the left: GFP signals of TNRC6A fragments; Cy5 signals of the second antibody against anti-Ago2 antibody; the merged images of GFP and Cy5, in which GFP is shown in green, the Cy5 in magenta, and the overlapped GFP/Cy5 in white; and DAPI signals. Bars, 5 μm. Protein expression was verified by Western blot (Supplemental Fig. S1C). (C) The ratio of cells expressing GFP exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow).
FIGURE 5.
FIGURE 5.
Nuclear TNRC6A is colocalized with miRNAs. (A) Cell lysates from HeLa cells expressing myc-GFP-TNRC6A and myc-GFP-TNRC6A-del.GW-I+II+III with or without NES mutation were immunoprecipitated with an anti-GFP antibody or rabbit normal IgG. Then, the immunoprecipitates (IP) and cell lysates were analyzed by Western blot using a monoclonal anti-Ago2 antibody (upper panel) and an anti-GFP antibody (lower panel). Asterisks indicate products of myc-GFP-TNRC6A and myc-GFP-TNRC6A-del.GW-I+II+III with or without NES mutation. (B) The amount of miRNAs contained in the immunoprecipitates was quantified by real-time PCR. Data are the mean and standard deviation of three independent experiments. (C–H) HeLa cells expressing myc-GFP-TNRC6A (WT; C,D), myc-GFP-TNRC6A-NES-mut (NES-mut; E,F), or myc-GFP-TNRC6A-del.GW-I+II+III-NES-mut (del.GW-I+II+III-NES-mut; G,H) were stained by digoxigenin-labeled LNA probe targeting miR-21 (C,E,G) or control probe (D,F,H). Fluorescent images are shown, from the left: GFP signals of TNRC6A; Rhodamine signals of the antibody against miRNA probes; the merged images of GFP and Rhodamine, in which GFP is shown in green and the Rhodamine in magenta; and DAPI signals. In the merged images, the dashed lines represent nuclear outlines. Bars, 10 μm.
FIGURE 6.
FIGURE 6.
miRNA-mediated gene silencing activity of TNRC6A with NES and/or NLS mutations. (A,B) TNRC6A (A) and TNRC6B (B) mRNA levels in HeLa cells transfected with siTNRC6A/siTNRC6B#1 or siTNRC6A/siTNRC6B#2. (C) One d after the transfection with siControl, siTNRC6A/siTNRC6B#1 or siTNRC6A/siTNRC6B#2, cells were further transfected with a mixture of the following four plasmids: psiCHECK-CXCR4 target, pGL3-Control, pSUPER-CXCR4 or control pSUPER-DsRed, and myc-GFP or the indicated TNRC6A expression constructs. Two d after second transfection, luciferase activities were measured. The activities were shown as the mean and standard deviation of three or four independent experiments. P-values were determined by Student's t-test. (*) P < 0.05. (D,E) Fluorescent microscopy images of the cells expressing WT (D) or NES-mut (E) under the same conditions shown in C. WT was exclusively localized in the cytoplasm, but NES-mut was in the nucleus. Bars, 20 μm. (F) Relative luciferase activities by the transfection of NES-mutated TNRC6As lacking various combinations of GW motifs.
FIGURE 7.
FIGURE 7.
Effects of overexpression of TNRC6A and its NES or NLS mutants on RNAi activity against MALAT-1. (A) The induction of FLAG-HA-SBP-TNRC6A or its mutant proteins by the treatment of the indicated concentration of dox for 24 h in HEK293 cells stably transfected with either empty vector, FLAG-HA-SBP-TNRC6A-WT, FLAG-HA-SBP-TNRC6A-NES-mut, or FLAG-HA-SBP-TNRC6A-NLS-mut expression constructs was verified by Western blot using anti-TNRC6A antibody. Anti-tubulin antibody was used as a loading control. (B–E) Stable HEK293 cells with pcDNA5/FRT/TO/FLAG-HA-SBP (empty vector; B), and expressing FLAG-HA-SBP-TNRC6A (C), FLAG-HA-SBP-TNRC6A-NES-mut (D), and FLAG-HA-SBP-TNRC6A-NLS-mut (E) were transfected with siControl2 or siMALAT-1 and cultured in the presence of the indicated concentration of dox for 24 h. MALAT-1 RNA levels were shown as the mean and standard deviation of three independent experiments. P-values were determined by Student's t-test. (*) P < 0.05.
FIGURE 8.
FIGURE 8.
Dcp1 and p54 were colocalized with cytoplasmic but not with nuclear TNRC6A. (A,C) HeLa cells transfected with pmyc-GFP-TNRC6A were treated with or without LMB, and stained with anti-Dcp1 (A) or anti-p54 (C) antibody. Fluorescent images are shown, from the left: GFP signals of TNRC6A; Cy5 signals of the second antibody against anti-Dcp1 (A) or anti-p54 (C) antibody; the merged images of GFP and Cy5, in which GFP is shown in green and the Cy5 in magenta; and DAPI signals. Bars, 10 μm. (B,D) HeLa cells transfected with pmyc-GFP-TNRC6A-NES-mut were stained with anti-Dcp1 (B) or anti-p54 (D) antibody. Fluorescent images are shown, from the left: GFP signals of TNRC6A; Cy5 signals of the second antibody against anti-Dcp1 (B) or anti-p54 (D) antibody; the merged images of GFP and Cy5, in which GFP is shown in green and the Cy5 in magenta; and DAPI signals. Bars, 10 μm.
FIGURE 9.
FIGURE 9.
TNRC6A regulates miRNA-mediated gene silencing in the nucleus and cytoplasm by navigating Ago protein. TNRC6A translocates from the cytoplasm to the nucleus via its own NLS sequence. Its nuclear export is mediated by Exportin 1 (XPO1), which directly binds to its NES sequence. In the cytoplasm, interaction between Ago and TNRC6A triggers translational repression and/or mRNA degradation by a known mechanism (see Introduction). In the nucleus, the miRNA-Ago-TNRC6A complex might induce miRNA-mediated gene silencing targeting nuclear RNA by an unknown mechanism. See the text for details.
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References

    1. Afonina E, Stauber R, Pavlakis GN 1998. The human poly(A)-binding protein 1 shuttles between the nucleus and the cytoplasm. J Biol Chem 273: 13015–13021 - PubMed
    1. Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E 2006. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20: 1885–1898 - PMC - PubMed
    1. Bohnsack MT, Czaplinski K, Gorlich D 2004. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10: 185–191 - PMC - PubMed
    1. Chekulaeva M, Filipowicz W, Parker R 2009. Multiple independent domains of dGW182 function in miRNA-mediated repression in Drosophila. RNA 15: 794–803 - PMC - PubMed
    1. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, et al. 2005. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Res 33: e179 doi: 10.1093/nar/gni178 - PMC - PubMed

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