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FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure.

Bensaid M, Melko M, Bechara EG, Davidovic L, Berretta A, Catania MV, Gecz J, Lalli E, Bardoni B - Nucleic Acids Res. (2009)

Bottom Line: We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity.Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced.All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6097-Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

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Endogenous and overexpressed FMR2 localizes to nuclear speckles. Co-localization of FMR2 with SC35 in nuclear speckles as detected by polyclonal anti-FMR2 antibody in Hela cells transfected with full-length FMR2 (A) and in NG108 cells (B) and in primary hippocampal neurons (C) expressing endogenous FMR2. FMR2 was detected with polyclonal anti-FMR2 antibody and SC35 was detected by monoclonal anti-SC35 antibody. (A) 63× magnification, scale bar 5 μm. (B and C) 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analyzed, showing a comparable result.
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Figure 1: Endogenous and overexpressed FMR2 localizes to nuclear speckles. Co-localization of FMR2 with SC35 in nuclear speckles as detected by polyclonal anti-FMR2 antibody in Hela cells transfected with full-length FMR2 (A) and in NG108 cells (B) and in primary hippocampal neurons (C) expressing endogenous FMR2. FMR2 was detected with polyclonal anti-FMR2 antibody and SC35 was detected by monoclonal anti-SC35 antibody. (A) 63× magnification, scale bar 5 μm. (B and C) 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analyzed, showing a comparable result.

Mentions: To get insight into FMR2 function, we performed a detailed study of its intracellular localization by generating a polyclonal anti-FMR2 antibody using a synthetic peptide corresponding to amino acids 116–133 of mouse FMR2 (see Supplementary Material). We then transfected HeLa cells (which do not express endogenous FMR2, Figure 1 and Supplementary Figure 1) with a construct encoding full-length FMR2 and we revealed its nuclear localization in a dot-like pattern (Figure 1A). This same result was obtained with a Flag-tagged-FMR2 (not shown). Our anti-FMR2 antibody recognizes endogenous FMR2 protein in neuroblastoma NG108 cells (Figure 1B) and in primary hippocampal neurons (Figure 1C). Intriguingly, both endogenous and transfected FMR2 are present in the nucleus in large dots colocalized with nuclear speckle domains, as revealed by the anti-SC35 monoclonal antibody (Figure 1, Supplementary Figure S2). These nuclear domains represent sites where splicing factors are concentrated, assembled and modified (25) (Supplementary Figure 2).Figure 1.


FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure.

Bensaid M, Melko M, Bechara EG, Davidovic L, Berretta A, Catania MV, Gecz J, Lalli E, Bardoni B - Nucleic Acids Res. (2009)

Endogenous and overexpressed FMR2 localizes to nuclear speckles. Co-localization of FMR2 with SC35 in nuclear speckles as detected by polyclonal anti-FMR2 antibody in Hela cells transfected with full-length FMR2 (A) and in NG108 cells (B) and in primary hippocampal neurons (C) expressing endogenous FMR2. FMR2 was detected with polyclonal anti-FMR2 antibody and SC35 was detected by monoclonal anti-SC35 antibody. (A) 63× magnification, scale bar 5 μm. (B and C) 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analyzed, showing a comparable result.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651778&req=5

Figure 1: Endogenous and overexpressed FMR2 localizes to nuclear speckles. Co-localization of FMR2 with SC35 in nuclear speckles as detected by polyclonal anti-FMR2 antibody in Hela cells transfected with full-length FMR2 (A) and in NG108 cells (B) and in primary hippocampal neurons (C) expressing endogenous FMR2. FMR2 was detected with polyclonal anti-FMR2 antibody and SC35 was detected by monoclonal anti-SC35 antibody. (A) 63× magnification, scale bar 5 μm. (B and C) 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analyzed, showing a comparable result.
Mentions: To get insight into FMR2 function, we performed a detailed study of its intracellular localization by generating a polyclonal anti-FMR2 antibody using a synthetic peptide corresponding to amino acids 116–133 of mouse FMR2 (see Supplementary Material). We then transfected HeLa cells (which do not express endogenous FMR2, Figure 1 and Supplementary Figure 1) with a construct encoding full-length FMR2 and we revealed its nuclear localization in a dot-like pattern (Figure 1A). This same result was obtained with a Flag-tagged-FMR2 (not shown). Our anti-FMR2 antibody recognizes endogenous FMR2 protein in neuroblastoma NG108 cells (Figure 1B) and in primary hippocampal neurons (Figure 1C). Intriguingly, both endogenous and transfected FMR2 are present in the nucleus in large dots colocalized with nuclear speckle domains, as revealed by the anti-SC35 monoclonal antibody (Figure 1, Supplementary Figure S2). These nuclear domains represent sites where splicing factors are concentrated, assembled and modified (25) (Supplementary Figure 2).Figure 1.

Bottom Line: We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity.Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced.All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6097-Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

Show MeSH
Related in: MedlinePlus