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First published online 24 July 2007
doi: 10.1242/jcs.009225

Journal of Cell Science 120, 2774-2784 (2007)
Published by The Company of Biologists 2007
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The exon-junction-complex-component metastatic lymph node 51 functions in stress-granule assembly

Aurélie Baguet1, Sébastien Degot1,*, Nicolas Cougot2, Edouard Bertrand2, Marie-Pierre Chenard3, Corinne Wendling1, Pascal Kessler1, Hervé Le Hir4, Marie-Christine Rio1 and Catherine Tomasetto1,{ddagger}

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie du Cancer, UMR 7104 CNRS/U596 INSERM/Université Louis Pasteur, BP 10142, 67404 Illkirch, C.U. de Strasbourg, France
2 Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535-IFR 24, 1919 route de Mende 34000 Montpellier, France
3 Departement de Pathologie Générale, Centre Hospitalier Universitaire de Hautepierre, 67098 Strasbourg, France
4 Centre de Génétique Moléculaire, CNRS UPR2167, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France


Figure 1
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  		Fig. 1. The endogenous MLN51 protein is recruited to SGs. (A) Immunofluorescence analysis of MLN51 and FMRP mobilization into SGs after arsenite treatment. HeLa cells, control (a-c, untreated) or treated with 0.5 mM arsenite for 1 hour (d-f) were either immediately fixed (a-f) or allowed to recover for 1 (g-i) or 2 (j-l) hours in normal medium without arsenite prior to fixation. Next, cells were co-labelled with anti-MLN51 (a,d,g,j, green) and anti-FMRP (b,e,h,k, red) antibodies. Nuclei were counterstained with Hoechst 33258 (blue) and corresponding merged images are shown on the right (c,f,i,l). (B) Immunofluorescence analysis of HeLa cells transiently transfected with the pEGFP-DCP1 plasmid (green). Control- and arsenite-treated cells were stained with an anti-MLN51 antibody (red), nuclei were counterstained with Hoechst 33258 (blue). Note that the endogenous MLN51 protein is not recruited into Dcp bodies.

 

Figure 2
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  		Fig. 2. SGs show discrete microdomains and dynamic shuttling of some components. (A) SGs are positive for MLN51, TIA1 and FMRP. Arsenite-treated HeLa cells, were fixed and co-labelled with anti-MLN51 (a, purple), anti-TIA1 (c, red) and anti-FMRP (b, green) antibodies. Nuclei were counterstained with Hoechst (d, blue) and the merged image of a, b and c is shown in e. (B) Confocal analysis of MLN51 and FMRP distribution into SGs. Arsenite-treated cells were co-labelled using anti-MLN51 (green) and anti-FMRP (red) antibodies and with Hoechst (blue). One section of 200 nm of the merge image is shown; inserts on the top left and lower right inside are higher magnification (2.5 fold) images of the SGs present in dotted squares. (C) FRAP analysis of MLN51Fl and MLN51Ct mobilization into SGs after arsenite treatment. HeLa cells transfected with EYFP-tagged MLN51 full length (fl) and MLN51/377-703 (Ct) and GFP-tagged hnRNPA1 and PABP for 48 hours and treated with arsenite were analysed by FRAP. One representative cell expressing EYFP-MLN51Fl (a) or EYFP-MLN51Ct (b) is shown. In both cases one granule (square boxes) was bleached and pictures were taken every 3 seconds. Time is indicated in seconds on each picture. (c) Graphical representation of fluorescence recovery patterns. Curves represent fluorescence intensity over time (seconds) and each curve corresponds to ten independent experiments.

 

Figure 3
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  		Fig. 3. Recruitment of MLN51 to SGs does not require its association with the exon junction complex. (A) Schematic representation of MLN51-derived constructs, indicating different consensus motifs including the SELOR module. The white and grey boxes show the position of the N- and C-terminal constructs, respectively. (B) Immunofluorescence analysis of HeLa cells transfected with full length and truncated EYFP-tagged MLN51 proteins (green). Arsenite-treated cells were fixed 24 hours, after transfection. (C) Immunofluorescence analysis of HeLa cells transfected with EYFP-tagged MAGOH (a,c, green) and Y14 (d,f, green). Arsenite-treated cells were stained with an anti-FMRP antibody (b,e, red). Nuclei were counterstained with Hoechst (blue).

 

Figure 4
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  		Fig. 4. Up- and downregulation of MLN51 in HeLa cells. (A) Western blot containing ~20 µg of total protein obtained from: HeLa Tet-On control cells (lanes 1 and 2) stably transfected with the empty pUHD vector; HeLa Tet-On MLN51Fl (lanes 3 and 4) and HeLa Tet-On MLN51Nt (lanes 5 and 6) cells, transfected with pUHD-MLN51/1-703 and pUHD-MLN51/1-383, respectively. The blot was probed with an anti-MLN51 against the SELOR domain of MLN51 and then probed with an anti-tubulin antibody which was used as a loading control. A 24-hour induction with doxycyclin is indicated on the top (+dox). The Arrow and arrowhead on the right indicate the position of MLN51Fl and MLN51Nt, respectively. (B) Western blot analysis of MLN51 depletion in HeLa cells. Western blot containing 20 µg of total protein obtained from HeLa cells transfected twice with either synthetic control siRNA (lanes 1 and 3) or MLN51-specific siRNA (lanes 2 and 4), and with pEYFP-MLN51ins plasmid was probed with anti-MLN51 and anti-tubulin (loading control) antibodies. The positions of the endogenous MLN51 and of the EYFP-MLN51 fusion protein are indicated on the right. (C) Analysis of arsenite-induced EIF2{alpha} (EIF2{alpha}) phosphorylation in the presence of MLN51Fl and MLN51Nt. 20 µg of total protein obtained from doxycyclin-induced HeLa Tet-On control (lanes 1 and 2); HeLa Tet-On MLN51Fl (lanes 3 and 4) and HeLa Tet-On MLN51Nt (lanes 5 and 6) cells, treated (+) or not (–) with arsenite, were analysed by western blotting using anti-MLN51-, anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies. Arrow and arrowhead on the right indicate the position of MLN51Fl and MLN51Nt, respectively. (D) Analysis of arsenite-induced EIF2{alpha} phosphorylation in MLN51-silenced cells. 20 µg of total protein obtained from HeLa cells transfected twice with either synthetic control siRNA (lanes 1 and 2) or MLN51-specific siRNA (lanes 3 and 4), treated (+) or not (–) with arsenite, were analysed by western blotting using anti-MLN51-, anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies. (E) Analysis of EIF2{alpha} phosphorylation in HeLa cells treated with arsenite or hippuristanol. 20 µg of total protein obtained from HeLa cells untreated (–, lane 1), treated with arsenite (A, lane 2) or with hippuristanol (H, lane 3) were analysed by western blotting using anti-EIF2{alpha}-, anti-phospho-EIF2{alpha}- and anti-tubulin-specific antibodies.

 

Figure 5
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  		Fig. 5. MLN51 downregulation alters SG assembly. (A) HeLa Tet-On MLN51Fl (a-l) and HeLa Tet-On MLN51Nt (m-x) were induced for 24 hours with doxycyclin and incubated in normal medium (untreated) or with arsenite for 30 minutes before fixation. Cells were double-stained with anti-MLN51 (green) and anti-FMRP (red) antibodies or with anti-MLN51 (green) antibody and a fluorescent oligo(dT) probe (red) to detect poly(A)+ RNA as indicated on the top. Note that cells expressing MLN51Nt treated with arsenite do not have FMRP or poly(A)+ RNA cytoplasmic foci compared with the non-induced cells present in the same fields (asterisks). (B) HeLa cells, transfected with Flag-tagged MLN51Nt (a-c), EYFP-tagged MLN51Nt (d-f, green) and as control EYFP-tagged MLN51Fl (g-i, green) plasmids, were treated with arsenite before fixation and probed with anti-Flag (a,c, green) and/or anti-FMRP (red, b,c,e,f,h,i) antibodies. Note that in the merged images (c,f), cells overexpressing Flag-MLN51Nt or EYFP-MLN51Nt have no FMRP-positive cytoplasmic foci (arrows). (C) HeLa control cells (a-c) were treated with hippuristanol before fixation and probed with anti-MLN51 (green, a,c) and anti-FMRP (red, b,c) antibodies. HeLa Tet-On MLN51Fl (d-f) and HeLa Tet-On MLN51Nt (g-i) were induced for 24 hours with doxycyclin and incubated with hippuristanol before fixation. Cells were double-stained with anti-MLN51 (green) and anti-FMRP (red) antibodies. Note that cells overexpressing MLN51Nt have no FMRP-positive cytoplasmic foci (arrows).

 

Figure 6
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  		Fig. 6. Depletion of MLN51 reduces cell viability after stress release. (A) Upper panel: HeLa cells were transfected twice with a MLN51-specific siRNA, treated with arsenite before fixation and double stained with anti-MLN51 (green) and anti-FMRP (red) antibodies. Cells showing undetectable level of MLN51 and no FMRP-positive foci are indicated by arrows and cells showing no depletion of MLN51 and FMRP-positive foci are indicated by asterisks. Bottom panel, MLN51-silenced HeLa cells were transfected using a siRNA-insensitive plasmid pEYFP-MLN51ins and treated with arsenite and stained with anti-MLN51 (purple) and anti-FMRP (red) antibodies. A cell showing undetectable level of MLN51 and no FMRP-positive foci is indicated by an arrow and a cell showing expression of EYPF-MLN51 and FMRP-positive foci is indicated by an asterisk. (B) Silencing of MLN51 alters SG formation. SGs were traced using an anti-FMRP antibody in arsenite-treated HeLa cells transfected with either synthetic control siRNA (siRNA/control, black bar) or MLN51-specific siRNA (siRNA/MLN51, white bar). The numbers of cells showing clear FMRP-positive foci were counted. Values presented are mean percentages ± s.e.m. of n=2 independent experiments done in triplicate, with *** indicating a P value of less than 0.001. (C) Depletion of MLN51 results in reduced cell viability following stress. HeLa cells were transfected with either control synthetic siRNA (black circles) or MLN51-specific siRNA (white circles). 48 hours after transfection, cells were stressed with sorbitol for 2.5 hours, followed by incubation in normal medium. Cellular viability was measured at each time point. Values presented are mean percentages ± s.e.m. of a typical experiment made in triplicate with ** and *** indicating a P value of less than 0.01 and 0.001, respectively. Top right: western blot analysis showing the level of MLN51 depletion in the experiment presented.

 

Figure 7
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  		Fig. 7. MLN51-overexpressing breast tumours contain cytoplasmic foci resembling SGs. (A) Immunohistochemical analysis of MLN51 subcellular localization in two ductal carcinomas. In cancer cells, MLN51 staining is either diffuse in the cytoplasm (a) or appears in punctate cytoplasmic structures (b). Sections are counterstained with Haematoxylin (nuclei are violet). (B) MLN51-expressing tumours were double-stained for MLN51 (green) and FMRP (red) or with MLN51 (green) and PABP (red). Nuclei were counterstained with Hoechst (blue). Note that both FMRP and PABP are present in cytoplasmic foci positive for MLN51. Bar, 10 µm.

 

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© The Company of Biologists Ltd 2007