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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 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. (A) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. (B) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (C) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (D) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.
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Figure 2: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. (A) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. (B) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (C) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (D) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

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.


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 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. (A) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. (B) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (C) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (D) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.
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Figure 2: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. (A) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. (B) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (C) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). (D) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.
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.

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