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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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FMR2 determinants for its intracellular localization. (A) Co-localization of C-ter with SC35 in nuclear speckles in Hela cells. (B) Localization of N-ter in the cytoplasm of HeLa cells. (C) Nucleolar localization of C1 in HeLa. All FMR2 domains have been detected using the polyclonal anti-Flag antibody, and SC35 was detected by the anti SC35 monoclonal antibody. The 40× magnification, scale bar 10 μm. Twenty 40× fields were analyzed, showing a comparable result.
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Figure 2: FMR2 determinants for its intracellular localization. (A) Co-localization of C-ter with SC35 in nuclear speckles in Hela cells. (B) Localization of N-ter in the cytoplasm of HeLa cells. (C) Nucleolar localization of C1 in HeLa. All FMR2 domains have been detected using the polyclonal anti-Flag antibody, and SC35 was detected by the anti SC35 monoclonal antibody. The 40× magnification, scale bar 10 μm. Twenty 40× fields were analyzed, showing a comparable result.

Mentions: To define the functional properties of FMR2 domains, we divided FMR2 into three regions and subcloned them fused to Flag-tag: N-ter (residues 1–640), C-ter (residues 633–1272), C1 (residues 633–966). We used these constructs to transfect HeLa cells and their protein products were revealed by immunofluorescence using the anti-Flag antibody. The N-ter construct is localized in the cytoplasm (Figure 2), while C-ter exhibits the same nuclear distribution as the full-length protein, co-localizing with SC35 and C1 is nucleolar (Figure 2) (in Supplementary Figure 2B the co-localization of C1 with fibrillarin as a nucleolar marker is shown). We conclude that the C-ter domain is determinant for the speckle localization of FMR2. Interestingly, this domain also contains a nucleolar localization signal in its C1 subdomain.Figure 2.


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)

FMR2 determinants for its intracellular localization. (A) Co-localization of C-ter with SC35 in nuclear speckles in Hela cells. (B) Localization of N-ter in the cytoplasm of HeLa cells. (C) Nucleolar localization of C1 in HeLa. All FMR2 domains have been detected using the polyclonal anti-Flag antibody, and SC35 was detected by the anti SC35 monoclonal antibody. The 40× magnification, scale bar 10 μm. Twenty 40× fields were analyzed, showing a comparable result.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: FMR2 determinants for its intracellular localization. (A) Co-localization of C-ter with SC35 in nuclear speckles in Hela cells. (B) Localization of N-ter in the cytoplasm of HeLa cells. (C) Nucleolar localization of C1 in HeLa. All FMR2 domains have been detected using the polyclonal anti-Flag antibody, and SC35 was detected by the anti SC35 monoclonal antibody. The 40× magnification, scale bar 10 μm. Twenty 40× fields were analyzed, showing a comparable result.
Mentions: To define the functional properties of FMR2 domains, we divided FMR2 into three regions and subcloned them fused to Flag-tag: N-ter (residues 1–640), C-ter (residues 633–1272), C1 (residues 633–966). We used these constructs to transfect HeLa cells and their protein products were revealed by immunofluorescence using the anti-Flag antibody. The N-ter construct is localized in the cytoplasm (Figure 2), while C-ter exhibits the same nuclear distribution as the full-length protein, co-localizing with SC35 and C1 is nucleolar (Figure 2) (in Supplementary Figure 2B the co-localization of C1 with fibrillarin as a nucleolar marker is shown). We conclude that the C-ter domain is determinant for the speckle localization of FMR2. Interestingly, this domain also contains a nucleolar localization signal in its C1 subdomain.Figure 2.

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