Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.
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.
Affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, 34295 Montpellier, France.Show MeSH
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Mentions: First, we carried out a Y2H screen using full-length human RBPMS2 as bait against a human placenta library. We isolated eight different clones that corresponded to six RBPMS2 sequences. All included a common RBPMS2 region within residues 32 and 105, indicating that the self-interaction region was located in the RRM domain (Figure 2A). We next examined whether RBPMS2 self-associated in vitro by co-immunoprecipitation assays (co-IP) using lysates of DF-1 cells that express HA-tagged human RBPMS2 or the small GTPase TC10 protein fused to the HA-tag (negative control) with or without Myc-tagged RBPMS2. HA-RBPMS2, but not HA-TC10, co-precipitated with Myc-RBPMS2 (Figure 2B). In addition, RNase treatment did not affect co-IP of HA-RBPMS2 and Myc-RBPMS2, indicating that this interaction is specific and direct and does not require any RNA molecule (Figure 2C). Then, by using the in situ PLA (DuoLink technology) we showed that in DF-1 cells that co-express Myc- and HA-RBPMS2, the interaction between HA-RBPMS2 and Myc-RBPMS2 occurred in the cytoplasm. As negative controls, we also tested and confirmed the absence of interaction between RBPMS2 with the unrelated Myc-NICD or HA-TC10 proteins (Figure 2D). These findings demonstrate that RBPMS2 is a subunit, which self-associates in the cytoplasm through the RRM domain and independently of any RNA.
Affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, 34295 Montpellier, France.