A Masked PY-NLS in Drosophila TIS11 and Its Mammalian Homolog Tristetraprolin
Figure 5
Screening of Drosophila importins to identify dTIS11 nuclear import machinery.
(A) Human karyopherins and their Drosophila homologs. The identity/similarity of sequence is indicated. (B) Inhibition of Drosophila karyopherin expression by RNA interference. S2 cells were incubated for 4 days in the absence or in the presence of dsRNA targeting the indicated karyopherins. Cells were harvested and karyopherin mRNAs were quantified by qRT-PCR. The values are expressed relatively to untreated samples. The error bars represents the standard deviation over a triplicate measure. (C) Subcellular distribution of Cherry-dTIS11 C179A protein upon inhibition of karyopherin expression. S2 cells were treated with dsRNA targeting the indicated karyopherins as described in (B), transfected with Cherry-dTIS11 C179A construct and treated with LMB (10 ng/ml, 1h). Data acquisition and quantification were performed as in Fig. 2. Bars show the average of the nuc/cyt ratio ± s.d. ns: non-significant (p>0.05); ***: p<0.0001 (U-tests). (D) Representative images of the nucleo-cytoplasmic distribution of Cherry-dTIS11 C179A mutant in Ketel dsRNA- and Trn dsRNA-treated S2 cells in the presence of LMB. Bar = 5 µm. Results shown in (B), (C) and (D) are representative of three independent experiments. (E) S2 cells were transfected to express wild-type or mutant versions of dTIS11 fused to Cherry, in combination with GFP alone or in fusion with the M9M peptide. Twenty hours post-transfection, cells were treated with LMB (10 ng/ml, 1h), fixed and observed by direct fluorescence microscopy. The subcellular distribution of the Cherry fusions was quantified as described in Fig. 2 for cells exhibiting GFP fluorescence. Bars show the average of the nuc/cyt ratio ± s.d. *: p<0.05; ***: p<0.0001; ns: non-significant (p>0.05) (t-tests). The absence of significant difference between Cherry-dTIS11 WT nuc/cyt ratios in GFP versus GFP-M9M-expressing cells and the significant difference between Cherry-dTIS11 C179A nuc/cyt ratios in these two conditions were observed in five independent experiments. (F) Representative images of the nucleo-cytoplasmic distribution of Cherry-dTIS11 C179A in S2 cells expressing GFP or GFP-M9M and treated with LMB as in (E). Bar = 5 µm.