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Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

Sagnol S, Yang Y, Bessin Y, Allemand F, Hapkova I, Notarnicola C, Guichou JF, Faure S, Labesse G, de Santa Barbara P - Nucleic Acids Res. (2014)

Bottom Line: RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins.We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action.Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, 34295 Montpellier, France.

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RBPMS2 homodimerization is necessary to dedifferentiate digestive SMCs. (A) Immunofluorescence analysis of primary SMC cultures infected with RCAS-empty, RCAS-Myc-RBPMS2 and RCAS-Myc-RBPMS2-L40E retroviruses for 3 days. Nuclei were visualized with Hoechst. Anti-Calponin antibodies were used as a marker of SMC differentiation and anti-Myc antibodies to identify cells infected by retroviruses that express chicken RBPMS2 or RBPMS2-L40E. (B) Quantification of mitotic cells using anti-phosphorylated Histone 3-Ser10 (PH3) antibodies in primary SMC cultures infected with retroviruses that express RBPMS2 or RBPMS2-L40E retroviruses or controls (RCAS-empty) for 7 days. Values are the mean ± standard error of the mean of two independent experiments (RCAS-RBPMS2 or RCAS-RBPMS2 L40E versus RCAS-empty). **for P < 0.01; n.s. for no statistically significant. (C) Schematic representation of the eEF2 clones isolated by Y2H screening with human RBPMS2 as bait. Human eEF2 harbors one elongation factor domain (EF2) and three U5 small nuclear ribonucleoprotein domains (snRNP). (D) Immunoprecipitation with mouse anti-HA antibodies of protein lysates from HEK293 cells that express human HA-RBPMS2, HA-TC10 or not. Co-immunoprecipitation of endogenous eEF2 was monitored by immunoblotting with rabbit anti-eEF2 antibodies (upper panel). The efficiency of immunoprecipitation of HA-RBPMS2 and HA-TC10 was monitored by immunoblotting with mouse anti-HA antibodies (middle panel). Lower panel: 10% of total protein extracts from cells of each condition monitored by immunoblotting with rabbit anti-eEF2 antibodies. (E) Analysis of the interaction of Myc-RBPMS2 with HA-eEF2 by Duolink PLA in HEK293 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-eEF2. HA-eEF2 was detected with anti-mouse HA antibodies (in red) and Myc-tagged proteins with anti-rabbit Myc antibodies (in green). Protein interactions were detected with Duolink PLA labeled in magenta. Bars, 50 μm.
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Figure 5: RBPMS2 homodimerization is necessary to dedifferentiate digestive SMCs. (A) Immunofluorescence analysis of primary SMC cultures infected with RCAS-empty, RCAS-Myc-RBPMS2 and RCAS-Myc-RBPMS2-L40E retroviruses for 3 days. Nuclei were visualized with Hoechst. Anti-Calponin antibodies were used as a marker of SMC differentiation and anti-Myc antibodies to identify cells infected by retroviruses that express chicken RBPMS2 or RBPMS2-L40E. (B) Quantification of mitotic cells using anti-phosphorylated Histone 3-Ser10 (PH3) antibodies in primary SMC cultures infected with retroviruses that express RBPMS2 or RBPMS2-L40E retroviruses or controls (RCAS-empty) for 7 days. Values are the mean ± standard error of the mean of two independent experiments (RCAS-RBPMS2 or RCAS-RBPMS2 L40E versus RCAS-empty). **for P < 0.01; n.s. for no statistically significant. (C) Schematic representation of the eEF2 clones isolated by Y2H screening with human RBPMS2 as bait. Human eEF2 harbors one elongation factor domain (EF2) and three U5 small nuclear ribonucleoprotein domains (snRNP). (D) Immunoprecipitation with mouse anti-HA antibodies of protein lysates from HEK293 cells that express human HA-RBPMS2, HA-TC10 or not. Co-immunoprecipitation of endogenous eEF2 was monitored by immunoblotting with rabbit anti-eEF2 antibodies (upper panel). The efficiency of immunoprecipitation of HA-RBPMS2 and HA-TC10 was monitored by immunoblotting with mouse anti-HA antibodies (middle panel). Lower panel: 10% of total protein extracts from cells of each condition monitored by immunoblotting with rabbit anti-eEF2 antibodies. (E) Analysis of the interaction of Myc-RBPMS2 with HA-eEF2 by Duolink PLA in HEK293 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-eEF2. HA-eEF2 was detected with anti-mouse HA antibodies (in red) and Myc-tagged proteins with anti-rabbit Myc antibodies (in green). Protein interactions were detected with Duolink PLA labeled in magenta. Bars, 50 μm.

Mentions: We then asked whether RBPMS2 homodimerization was required also for regulating SMC plasticity. To this aim, we established primary cultures of digestive differentiated SMCs in serum-free medium supplemented with insulin and BSA as previously published (9). After 3 days, in control SMC cultures, Calponin, an SMC contractile marker, was homogeneously expressed in highly organized filament bundles and cells were spindle-shaped (Figure 5A; Supplementary Figure S6). Conversely, in cells that express wild-type RBPMS2, Calponin expression was lost. Interestingly, SMCs expressing RBPMS2-L40E present no decrease of Calponin expression compared to control (Figure 5A). Moreover, after 6 days of culture, the number of cells that expressed phosphorylated histone 3-Ser10 (PH3), a standard marker of G2/M transition, was 4.5-fold higher in cultures that overexpress wild-type RBPMS2 than in control cells and SMC cells that express RBPMS2-L40E (Figure 5B), as also observed in heterologous DF1 cell line (Figure 4D). In addition, we also compared the impact of human RBPMS2, RBPMS2-L49E and L49Q in primary SMC culture and found that RBPMS2-L49Q like RBPMS2 induced an alteration of Calponin expression, whereas RBPMS2-L49E not (Supplementary Figure S3B). These results demonstrate that RBPMS2 homodimerization is essential to induce SMC dedifferentiation and proliferation.


Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

Sagnol S, Yang Y, Bessin Y, Allemand F, Hapkova I, Notarnicola C, Guichou JF, Faure S, Labesse G, de Santa Barbara P - Nucleic Acids Res. (2014)

RBPMS2 homodimerization is necessary to dedifferentiate digestive SMCs. (A) Immunofluorescence analysis of primary SMC cultures infected with RCAS-empty, RCAS-Myc-RBPMS2 and RCAS-Myc-RBPMS2-L40E retroviruses for 3 days. Nuclei were visualized with Hoechst. Anti-Calponin antibodies were used as a marker of SMC differentiation and anti-Myc antibodies to identify cells infected by retroviruses that express chicken RBPMS2 or RBPMS2-L40E. (B) Quantification of mitotic cells using anti-phosphorylated Histone 3-Ser10 (PH3) antibodies in primary SMC cultures infected with retroviruses that express RBPMS2 or RBPMS2-L40E retroviruses or controls (RCAS-empty) for 7 days. Values are the mean ± standard error of the mean of two independent experiments (RCAS-RBPMS2 or RCAS-RBPMS2 L40E versus RCAS-empty). **for P < 0.01; n.s. for no statistically significant. (C) Schematic representation of the eEF2 clones isolated by Y2H screening with human RBPMS2 as bait. Human eEF2 harbors one elongation factor domain (EF2) and three U5 small nuclear ribonucleoprotein domains (snRNP). (D) Immunoprecipitation with mouse anti-HA antibodies of protein lysates from HEK293 cells that express human HA-RBPMS2, HA-TC10 or not. Co-immunoprecipitation of endogenous eEF2 was monitored by immunoblotting with rabbit anti-eEF2 antibodies (upper panel). The efficiency of immunoprecipitation of HA-RBPMS2 and HA-TC10 was monitored by immunoblotting with mouse anti-HA antibodies (middle panel). Lower panel: 10% of total protein extracts from cells of each condition monitored by immunoblotting with rabbit anti-eEF2 antibodies. (E) Analysis of the interaction of Myc-RBPMS2 with HA-eEF2 by Duolink PLA in HEK293 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-eEF2. HA-eEF2 was detected with anti-mouse HA antibodies (in red) and Myc-tagged proteins with anti-rabbit Myc antibodies (in green). Protein interactions were detected with Duolink PLA labeled in magenta. Bars, 50 μm.
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Figure 5: RBPMS2 homodimerization is necessary to dedifferentiate digestive SMCs. (A) Immunofluorescence analysis of primary SMC cultures infected with RCAS-empty, RCAS-Myc-RBPMS2 and RCAS-Myc-RBPMS2-L40E retroviruses for 3 days. Nuclei were visualized with Hoechst. Anti-Calponin antibodies were used as a marker of SMC differentiation and anti-Myc antibodies to identify cells infected by retroviruses that express chicken RBPMS2 or RBPMS2-L40E. (B) Quantification of mitotic cells using anti-phosphorylated Histone 3-Ser10 (PH3) antibodies in primary SMC cultures infected with retroviruses that express RBPMS2 or RBPMS2-L40E retroviruses or controls (RCAS-empty) for 7 days. Values are the mean ± standard error of the mean of two independent experiments (RCAS-RBPMS2 or RCAS-RBPMS2 L40E versus RCAS-empty). **for P < 0.01; n.s. for no statistically significant. (C) Schematic representation of the eEF2 clones isolated by Y2H screening with human RBPMS2 as bait. Human eEF2 harbors one elongation factor domain (EF2) and three U5 small nuclear ribonucleoprotein domains (snRNP). (D) Immunoprecipitation with mouse anti-HA antibodies of protein lysates from HEK293 cells that express human HA-RBPMS2, HA-TC10 or not. Co-immunoprecipitation of endogenous eEF2 was monitored by immunoblotting with rabbit anti-eEF2 antibodies (upper panel). The efficiency of immunoprecipitation of HA-RBPMS2 and HA-TC10 was monitored by immunoblotting with mouse anti-HA antibodies (middle panel). Lower panel: 10% of total protein extracts from cells of each condition monitored by immunoblotting with rabbit anti-eEF2 antibodies. (E) Analysis of the interaction of Myc-RBPMS2 with HA-eEF2 by Duolink PLA in HEK293 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-eEF2. HA-eEF2 was detected with anti-mouse HA antibodies (in red) and Myc-tagged proteins with anti-rabbit Myc antibodies (in green). Protein interactions were detected with Duolink PLA labeled in magenta. Bars, 50 μm.
Mentions: We then asked whether RBPMS2 homodimerization was required also for regulating SMC plasticity. To this aim, we established primary cultures of digestive differentiated SMCs in serum-free medium supplemented with insulin and BSA as previously published (9). After 3 days, in control SMC cultures, Calponin, an SMC contractile marker, was homogeneously expressed in highly organized filament bundles and cells were spindle-shaped (Figure 5A; Supplementary Figure S6). Conversely, in cells that express wild-type RBPMS2, Calponin expression was lost. Interestingly, SMCs expressing RBPMS2-L40E present no decrease of Calponin expression compared to control (Figure 5A). Moreover, after 6 days of culture, the number of cells that expressed phosphorylated histone 3-Ser10 (PH3), a standard marker of G2/M transition, was 4.5-fold higher in cultures that overexpress wild-type RBPMS2 than in control cells and SMC cells that express RBPMS2-L40E (Figure 5B), as also observed in heterologous DF1 cell line (Figure 4D). In addition, we also compared the impact of human RBPMS2, RBPMS2-L49E and L49Q in primary SMC culture and found that RBPMS2-L49Q like RBPMS2 induced an alteration of Calponin expression, whereas RBPMS2-L49E not (Supplementary Figure S3B). These results demonstrate that RBPMS2 homodimerization is essential to induce SMC dedifferentiation and proliferation.

Bottom Line: RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins.We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action.Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, 34295 Montpellier, France.

Show MeSH
Related in: MedlinePlus