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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 required for RBPMS2 function. (A) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP alone (negative control) or of RCAS-RBPMS2 and RCAS-GFP. The presence of retroviruses was confirmed by direct observation of GFP expression. Sustained expression of chicken RBPMS2 leads to specific stomach malformations: hypertrophied proventriculus (arrows) and denser and malformed gizzard (arrowheads). (B) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control) or of the RCAS-RBPMS2-L40E mutant that cannot homodimerize with RCAS-GFP. White arrows and arrowheads indicate, respectively, the gizzard and the proventriculus. (C) Whole-mount in situ hybridization analysis of NOGGIN expression in stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control), RCAS-RBPMS2 and RCAS-GFP, or RCAS-RBPMS2-L40E and RCAS-GFP. Infection was confirmed by direct observation of GFP expression. (D) Glutaraldehyde crosslink assay of protein extract from DF1 cells infected either with RCAS-GFP (positive control, left panel), RCAS-Myc-RBPMS2 or RCAS-Myc-RBPMS2-L40E (right panels), followed by SDS-PAGE separation and revealed by rabbit anti-GFP or anti-Myc antibodies. Anti-GFP antibody revealed GFP monomers and dimers (near 55 kDa, black arrow), as expected. Anti-Myc antibody revealed Myc-RBPMS2 monomers at 27 kDa on both Myc-RBPMS2 and Myc-RBPMS2-L40E expressing cell protein extract, but only Myc-RBPMS2 expressing cell protein extract presents Myc-RBPMS2 dimerization at 55 kDa (red arrow). Note the below 50-kDa bands (asterisk) on Myc-RBPMS2 expressing cell protein extract correspond to the induction of endogenous avian MYC protein.
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Figure 4: RBPMS2 homodimerization is required for RBPMS2 function. (A) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP alone (negative control) or of RCAS-RBPMS2 and RCAS-GFP. The presence of retroviruses was confirmed by direct observation of GFP expression. Sustained expression of chicken RBPMS2 leads to specific stomach malformations: hypertrophied proventriculus (arrows) and denser and malformed gizzard (arrowheads). (B) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control) or of the RCAS-RBPMS2-L40E mutant that cannot homodimerize with RCAS-GFP. White arrows and arrowheads indicate, respectively, the gizzard and the proventriculus. (C) Whole-mount in situ hybridization analysis of NOGGIN expression in stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control), RCAS-RBPMS2 and RCAS-GFP, or RCAS-RBPMS2-L40E and RCAS-GFP. Infection was confirmed by direct observation of GFP expression. (D) Glutaraldehyde crosslink assay of protein extract from DF1 cells infected either with RCAS-GFP (positive control, left panel), RCAS-Myc-RBPMS2 or RCAS-Myc-RBPMS2-L40E (right panels), followed by SDS-PAGE separation and revealed by rabbit anti-GFP or anti-Myc antibodies. Anti-GFP antibody revealed GFP monomers and dimers (near 55 kDa, black arrow), as expected. Anti-Myc antibody revealed Myc-RBPMS2 monomers at 27 kDa on both Myc-RBPMS2 and Myc-RBPMS2-L40E expressing cell protein extract, but only Myc-RBPMS2 expressing cell protein extract presents Myc-RBPMS2 dimerization at 55 kDa (red arrow). Note the below 50-kDa bands (asterisk) on Myc-RBPMS2 expressing cell protein extract correspond to the induction of endogenous avian MYC protein.

Mentions: To further investigate whether RBPMS2 homodimerization has a role in its biological function, we expressed chick RBPMS2 or the chick mutant of human RBPMS2-L49E (RBPMS2-L40E) in the gastrointestinal mesenchyme of stage-10 chicken embryos using an avian replication-competent retroviral misexpression system (10,18). In accordance with our previous results (9), sustained RBPMS2 expression resulted in a dramatic alteration of the stomach morphology: the proventriculus, which is the glandular part of the chick stomach, was hypertrophied and the gizzard was denser and malformed in comparison to controls that overexpressed GFP alone (Figure 4A). In contrast, sustained expression of RBPMS2-L40E did not induce any morphological change (n = 26, stomachs; Figure 4B). Moreover, while misexpression of wild-type RBPMS2 in the gastrointestinal mesenchyme upregulated NOGGIN mRNA expression in comparison to controls (Figure 4C, left and middle panels), misexpression of RBPMS2-L40E did not (n = 17 infected stomachs; Figure 4C, right panel). Lastly, we verified the homodimerization status of Myc-RBPMS2, Myc-RBPMS2-L40E and GFP proteins in DF1 cell line by glutaraldehyde crosslinking. Interestingly, whereas fixed protein extract from GFP (as positive control) or Myc-RBPMS2 expressing cells displays well-defined homodimeric forms, no clear dimerization was observed in fixed protein extract from Myc-RBPMS2-L40 expressing cells (Figure 4D). Altogether these results demonstrate that RBPMS2 homodimerization is essential for RBPMS2-mediated upregulation of NOGGIN.


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 required for RBPMS2 function. (A) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP alone (negative control) or of RCAS-RBPMS2 and RCAS-GFP. The presence of retroviruses was confirmed by direct observation of GFP expression. Sustained expression of chicken RBPMS2 leads to specific stomach malformations: hypertrophied proventriculus (arrows) and denser and malformed gizzard (arrowheads). (B) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control) or of the RCAS-RBPMS2-L40E mutant that cannot homodimerize with RCAS-GFP. White arrows and arrowheads indicate, respectively, the gizzard and the proventriculus. (C) Whole-mount in situ hybridization analysis of NOGGIN expression in stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control), RCAS-RBPMS2 and RCAS-GFP, or RCAS-RBPMS2-L40E and RCAS-GFP. Infection was confirmed by direct observation of GFP expression. (D) Glutaraldehyde crosslink assay of protein extract from DF1 cells infected either with RCAS-GFP (positive control, left panel), RCAS-Myc-RBPMS2 or RCAS-Myc-RBPMS2-L40E (right panels), followed by SDS-PAGE separation and revealed by rabbit anti-GFP or anti-Myc antibodies. Anti-GFP antibody revealed GFP monomers and dimers (near 55 kDa, black arrow), as expected. Anti-Myc antibody revealed Myc-RBPMS2 monomers at 27 kDa on both Myc-RBPMS2 and Myc-RBPMS2-L40E expressing cell protein extract, but only Myc-RBPMS2 expressing cell protein extract presents Myc-RBPMS2 dimerization at 55 kDa (red arrow). Note the below 50-kDa bands (asterisk) on Myc-RBPMS2 expressing cell protein extract correspond to the induction of endogenous avian MYC protein.
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Figure 4: RBPMS2 homodimerization is required for RBPMS2 function. (A) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP alone (negative control) or of RCAS-RBPMS2 and RCAS-GFP. The presence of retroviruses was confirmed by direct observation of GFP expression. Sustained expression of chicken RBPMS2 leads to specific stomach malformations: hypertrophied proventriculus (arrows) and denser and malformed gizzard (arrowheads). (B) Stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control) or of the RCAS-RBPMS2-L40E mutant that cannot homodimerize with RCAS-GFP. White arrows and arrowheads indicate, respectively, the gizzard and the proventriculus. (C) Whole-mount in situ hybridization analysis of NOGGIN expression in stomachs from E9 chicken embryos after retroviral misexpression of RCAS-GFP (control), RCAS-RBPMS2 and RCAS-GFP, or RCAS-RBPMS2-L40E and RCAS-GFP. Infection was confirmed by direct observation of GFP expression. (D) Glutaraldehyde crosslink assay of protein extract from DF1 cells infected either with RCAS-GFP (positive control, left panel), RCAS-Myc-RBPMS2 or RCAS-Myc-RBPMS2-L40E (right panels), followed by SDS-PAGE separation and revealed by rabbit anti-GFP or anti-Myc antibodies. Anti-GFP antibody revealed GFP monomers and dimers (near 55 kDa, black arrow), as expected. Anti-Myc antibody revealed Myc-RBPMS2 monomers at 27 kDa on both Myc-RBPMS2 and Myc-RBPMS2-L40E expressing cell protein extract, but only Myc-RBPMS2 expressing cell protein extract presents Myc-RBPMS2 dimerization at 55 kDa (red arrow). Note the below 50-kDa bands (asterisk) on Myc-RBPMS2 expressing cell protein extract correspond to the induction of endogenous avian MYC protein.
Mentions: To further investigate whether RBPMS2 homodimerization has a role in its biological function, we expressed chick RBPMS2 or the chick mutant of human RBPMS2-L49E (RBPMS2-L40E) in the gastrointestinal mesenchyme of stage-10 chicken embryos using an avian replication-competent retroviral misexpression system (10,18). In accordance with our previous results (9), sustained RBPMS2 expression resulted in a dramatic alteration of the stomach morphology: the proventriculus, which is the glandular part of the chick stomach, was hypertrophied and the gizzard was denser and malformed in comparison to controls that overexpressed GFP alone (Figure 4A). In contrast, sustained expression of RBPMS2-L40E did not induce any morphological change (n = 26, stomachs; Figure 4B). Moreover, while misexpression of wild-type RBPMS2 in the gastrointestinal mesenchyme upregulated NOGGIN mRNA expression in comparison to controls (Figure 4C, left and middle panels), misexpression of RBPMS2-L40E did not (n = 17 infected stomachs; Figure 4C, right panel). Lastly, we verified the homodimerization status of Myc-RBPMS2, Myc-RBPMS2-L40E and GFP proteins in DF1 cell line by glutaraldehyde crosslinking. Interestingly, whereas fixed protein extract from GFP (as positive control) or Myc-RBPMS2 expressing cells displays well-defined homodimeric forms, no clear dimerization was observed in fixed protein extract from Myc-RBPMS2-L40 expressing cells (Figure 4D). Altogether these results demonstrate that RBPMS2 homodimerization is essential for RBPMS2-mediated upregulation of NOGGIN.

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