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First published online December 3, 2008
doi: 10.1242/10.1242/jcs.036905

Journal of Cell Science 121, 4134-4144 (2008)
Published by The Company of Biologists 2008
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Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing

Songqing Li1, Shang L. Lian1,*, Joanna J. Moser2,*, Mark L. Fritzler2, Marvin J. Fritzler2, Minoru Satoh3 and Edward K. L. Chan1,{ddagger}

1 Departments of Oral Biology and Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
2 Department of Biochemistry and Molecular Biology, University of Calgary, Alberta T2N 4N1, Canada
3 Division of Rheumatology and Clinical Immunology, Department of Medicine and Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA


Figure 1
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  		Fig. 1. Schematic of the human TNRC6A gene products GW182 and TNGW1. (A) GW182 has been reported to be a 182-kDa protein product of 1709 amino acids and containing three GW-rich regions, a QN-rich region and a classical RNA recognition motif (RRM). TNGW1 is the novel 210-kDa GW182 isoform with an extra N-terminal 253-amino-acid polypeptide containing a stretch of glutamine-repeat (Q-repeat) that is translated from the CAG TNR. (B) TNRC6A resides in human chromosome 16p11.2. The 5' end corresponding to the mRNA of these two isoforms separates ~60 kb on chromosome 16. (C) The TNR Q-repeat domain was encoded from the fifth exon and the corresponding nucleotide and amino acid sequences are shown in this panel. (D) Alignment of N-terminal TNGW1 sequences from mouse, rat and human using ClustalW. The result shows sequence conservation within these three species with some degree of diversity (yellow) in the TNR Q-repeat region.

 

Figure 2
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  		Fig. 2. TNGW1 mRNA containing the TNR exon detected in human testis and different cell lines using RT-PCR. The primer set that flank the TNR region of TNGW1 mRNA (nucleotides 362-1626) amplified 1.2 kb bands from cDNA samples of human testis tissue, HEp-2, HeLa and HepG2 cells. The amplified 1.2 kb products from HEp-2, HeLa and human testis tissue were subsequently purified and their consensuses to reference sequence were confirmed by direct DNA sequencing.

 

Figure 3
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  		Fig. 3. Production and characterization of polyclonal and monoclonal antibodies specific to the TNGW1 isoform. (A) Rabbit polyclonal anti-rTNR antibodies strongly reacted with rTNR polypeptide coated in ALBIA. Compared with two rabbit anti-GW182 antibodies (5182 and 6642) and the pre-immune rabbit sera, rabbit anti-rTNR sera 6225 and 6226 strongly reacted with rTNR-coated beads. (B) Rabbit anti-rTNR antibodies recognized only TNGW1 but not GW182 in western blots. HeLa cells transfected with EGFP-tagged TNGW1, GW182, rTNR and EGFP were harvested 24 hours after transfection. These cell lysates, together with recombinant 6xHis-tagged rTNR, were analyzed by western blotting using rabbit anti-rTNR sera 6225 and 6226. The membranes shown in columns a and c were first blotted with rabbit anti-rTNR antibodies, stripped and then re-blotted with anti-EGFP antibodies to confirm the recombinant proteins were expressed (b and d, respectively). (C) Reactivity of mouse anti-rTNR mAb with rTNR polypeptide by using ALBIA. Three mouse mAbs (2E11, 5C8 and 2F11) showed a high number of MFUs to rTNR, whereas control mAb anti-GW182 4B6, anti-GW2-25 and culture medium showed little or no reactivity. Note that mAbs 2E11 and 5C8 showed an ~20-fold higher number of MFUs than 2F11. (D) Peptide array mapping of antibody reactivity to the TNR region. Two duplicate peptide array membranes containing 59 sequential 15-amino-acid peptides, each with 5-amino-acid offset spanning the TNR region were synthesized and used to examine antibody specificity. Membrane number 1 (#1, left) was probed sequentially using anti-EEA1control mAb, anti-rTNR mAbs 2E11 and 2F11, and rabbit anti-rTNR 6225. Membrane number 2 (#2, right) was probed sequentially using anti-GW182 mAb 4B6, anti-rTNR mAb 5C8, rabbit anti-rTNR 6226 and human serum 18033. Boxed areas show the peptide strings that reside in TNR Q-repeat domains (as indicated by underlined residues in Fig. 1C). All rabbit and human sera showed reactivity with multiple peptides. The three mouse anti-rTNR mAbs recognized a relatively narrow region outside of the TNR Q-repeat domain.

 

Figure 4
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  		Fig. 4. TNGW1 and GW182 are independent products of human TNRC6A gene. (A) Expression of both TNGW1 and GW182 were detected in HeLa cells. GWB components were enriched by IP using human anti-GWB serum 18033 and analyzed by western blotting using rabbit anti-GW182 antibodies 5182 and 6642, mouse anti-GW182 mAb 4B6 and anti-rTNR mAb 2E11. All three anti-GW182 antibodies recognized two bands of ~210 and ~180 kDa, whereas anti-rTNR 2E11 only recognized the 210-kDa band regarded as TNGW1 – the novel isoform of GW182. (B) TNGW1 was detected by multiple anti-rTNR antibodies. IP samples from serum18033 were also examined by rabbit polyclonal antibodies 6225, 6226, and mouse mAb 5C8 recognizing the N-terminal domain rTNR. A 210-kDa band was detected by each of these antibodies regarded as TNGW1. (C) TNGW1 knockdown using siRNA specifically targeting TNGW1 did not affect the level of GW182 in HeLa cells. HeLa cells were transfected with siRNA specifically targeting the TNGW1 isoform (siTNR) or siRNA specifically targeting both isoforms (siGW182) using lipofectamine 2000 (LP 2000). After 48 hours of transfection, cells were lysed and analyzed by western blotting using rabbit anti-rTNR serum 6225 or anti-GW182 serum 5182. Compared with mock transfected and untreated control groups, siGW182 knocked down both TNGW1 and GW182, whereas siTNR only knocked down TNGW1.

 

Figure 5
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  		Fig. 5. TNGW1 resided in a subset of GWBs. (A) Mouse monoclonal anti-rTNR antibody 2F11 stained a subset of GWBs that were recognized by the prototype serum 18033. HEp-2 cells were stained using mouse mAb 2F11, which stained a subset of GWBs that were detected by serum 18033 (arrows) but did not recognize other GWBs (arrowheads). Culture medium was used as a negative control. Bar, 10 µm. (B) Mouse anti-rTNR mAb 2F11 stained a subset of GWBs that were recognized by anti-GW182 mAb 4B6. HEp-2 cell staining was performed similarly using anti-rTNR 2F11 (IgG2a) and anti-GW182 4B6 (IgG1) and different fluorochrome-conjugated mouse IgG subclass-specific secondary antibodies to evaluate the distribution of TNGW1 in GW182-positive GWBs. Anti-rTNR 2F11 stained some of the foci detected by 4B6 (arrows) but not others (arrowhead). The second and third row of images show controls that had been incubated with either 4B6 or 2F11 and were stained with both secondary antibodies to demonstrate secondary antibody specificities. The asterisks in the first panel shows nonspecific nucleolar staining detected by the anti-IgG2a secondary antibodies. Bar 10 µm.

 

Figure 6
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  		Fig. 6. Intracellular localization of TNGW1 with other GWB components. (A) Both TNGW1 and GW182 colocalized to GWBs together with transfected Ago2. HeLa cells transfected with either GST-tagged GW182 or GST-tagged TNGW1 (red) and EGFP-Ago2 (green) were fixed and analyzed by indirect immunofluorescence 24 hours after transfection. Both TNGW1 and GW182 formed cytoplasmic foci and colocalized with Ago2. Bar, 10 µm. (B) Transfected TNGW1 colocalized with RNA decay factors enriched in GWBs. HeLa cells transfected with EGFP-TNGW1 were fixed 24 hours after transfection and analyzed by indirect immunofluorescence assay using rabbit anti-Dcp1a (red) and human serum IC6 (magenta, recognizes Ge-1, RAP55 and unrelated nuclear envelope protein). The transfected EGFP-TNGW1 formed cytoplasmic foci that were co-stained using both anti-Dcp1a and IC6 antibodies. Bar, 10 µm.

 

Figure 7
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  		Fig. 7. Knockdown of TNGW1 has no apparent effect on the assembly of GWBs in HeLa cells. HeLa cells were transfected with siTNR or siGW182 and then harvested at 0, 1, 2 or 3 days after transfection. Indirect immunofluorescence assay was performed by using rabbit anti-Dcp1a (red) and human anti-GWB serum 18033 (green) to examine the effect of either siRNA on GWB formation. Bar, 10 µm.

 

Figure 8
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  		Fig. 8. Tethered TNGW1 and GW182 exerted translational repression to a greater extent than Ago2. (A) Tethered TNGW1 and GW182 both exerted stronger repression effect than tethered Ago2. Assays for both luciferase activities were performed 48 hours after transfection. Tethered TNGW1 and GW182 showed 67.6% and 65.3% translation-repression effect to FL-5BoxB reporter, which was significantly higher than tethered Ago2 (*P<0.05, t-test). Measured FL-5BoxB activities were normalized to corresponding RL activities. All translation-repression effects were estimated by the differences of FL-5BoxB/RL activity compared with that in NHA-vector-transfected group. Error bars represent standard deviations. (B) Tethered TNGW1 or GW182 induced less reporter-mRNA-reduction than tethered Ago2. The mRNA levels from the same assay were measured by SYBR-Green quantitative RT-PCR. Degradation of FL-5BoxB mRNA were determined by the reductions of the mRNA level between experimental groups and NHA-vector transfected group. All FL-5BoxB mRNA levels were normalized to Renilla luciferase mRNA to minimize the experimental errors. (C) Tethered TNGW1 and GW182 strongly reduced the translation efficiency of the reporter compared with tethered Ago2. Translation efficiencies of FL-5BoxB in different groups were calculated by the ratio of relative FL-5BoxB activities to their mRNA levels. In HEK 293 cells, tethered Ago2 did not significantly alter the FL-5BoxB reporter translation efficiency. However, tethered TNGW1 and GW182 reduced the translation efficiency FL-5BoxB reporter by 57.5% and 54.0%, respectively (*P<0.01, t-test). Error bars represent standard deviations.

 

Figure 9
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  		Fig. 9. Translational repression of tethered Ago2 required endogenous TNGW1 and/or GW182. (A) The repression effect of tethered Ago2 was abolished when TNGW1 and GW182 were knocked down, whereas tethered TNGW1 or GW182 maintained repression in the absence of Ago2. HeLa cells were transfected with different siRNA 24 hours prior to the transfection of NHA-tagged constructs and RL-5BoxB/FL reporters. The translation-repression effect related to tethered Ago2, TNGW1 or GW182 was determined by reduction of RL activity and the repression effect in siRNA to EGFP (siGFP) transfected group was normalized to 1. The repression effect caused by NHA-Ago2 was abolished when cells were treated with siGW182 (asterisk, P<0.01, t-test). Knocked down of Ago2 (siAgo2) caused no significant effect on tethered GW182 or TNGW1 induced translational repression. Error bars represent standard deviations. (B) Tethered Ago2-mediated gene silencing required TNGW1 and/or GW182 regardless whether FL-5BoxB or RL-5BoxB was used as targeted reporter. Similar experiments were performed as described previously except for the use of RL-5BoxB reporter together with FL reporter as co-transfection quantitative control. The repression effect of tethered Ago2 was abolished consistently in the abscence of TNGW1 and/or GW182 when FL-5BoxB or RL-5BoxB were used as reporters, whereas tethered GW182 maintained its repression to both reporters in the absence of Ago2.

 

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© The Company of Biologists Ltd 2008