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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 is an RNA-binding protein.(A) In vitro translated [35S] methionine labelled full-length FMR2, full-length FMRP, N-ter and C-ter proteins were incubated with each RNA homopolymer linked to agarose in the presence of 0.25 M KCl. The same volume (10 μl) of each eluate was analysed by SDS–PAGE followed by fluorography. (B) Labelled N19 probe was incubated in the presence of increasing amounts of recombinant N-ter (lanes 2–4: 0.2, 0.4 and 0.6 pmol, respectively) and C-ter (lanes 5–7: 0.2, 0.4 and 0.6 pmol, respectively) proteins. As a control, the labelled N19 probe was shown in lane 1.
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Figure 4: FMR2 is an RNA-binding protein.(A) In vitro translated [35S] methionine labelled full-length FMR2, full-length FMRP, N-ter and C-ter proteins were incubated with each RNA homopolymer linked to agarose in the presence of 0.25 M KCl. The same volume (10 μl) of each eluate was analysed by SDS–PAGE followed by fluorography. (B) Labelled N19 probe was incubated in the presence of increasing amounts of recombinant N-ter (lanes 2–4: 0.2, 0.4 and 0.6 pmol, respectively) and C-ter (lanes 5–7: 0.2, 0.4 and 0.6 pmol, respectively) proteins. As a control, the labelled N19 probe was shown in lane 1.

Mentions: To validate our hypothesis, we tested the ability of FMR2 to bind synthetic RNA homopolymers. Indeed, FMR2 selectively binds to poly (G), but not poly (A), poly (C) or poly (U) (Figure 4A) while the control FMRP (Fragile X Mental Retardation Protein), as expected, is able to bind to poly (G) and poly (U) (20). The binding activity to poly (G) was retained by the FMR2 C-ter domain, the same region directing its localization in nuclear speckles (Figure 2), but not by the N-ter domain (Figure 2). Even if no homology with other RNA-binding domains is present in FMR2, two putative RNA-binding motifs were identified within this region (the first between residues 787 and 815 and the second between residues 890 and 917) using the RNABindR program (27). Since FMR2 has affinity for poly (G) RNA, we questioned whether it can bind any RNA containing a stretch of G nucleotides or if it is able to recognize and bind a specific G-rich RNA structure. For this reason, we tested the ability of N-ter and C-ter recombinant FMR2 proteins to bind a RNA probe containing a G-quartet forming structure, the N-19 RNA in a gel retardation assay. Previously, we have shown that FMR1 mRNA contains a G-quartet forming structure, the N19 sequence, localized between nucleotides 1470 and 1896 of FMR1, which is bound by FMRP itself (18,28). Indeed, as for the poly G-sequence increasing amounts of C-ter bind the N19 probe while of N-ter does not interact with the RNA (Figure 4B), suggesting a specificity of FMR2 for G-quartet structure via its C-terminal domain.Figure 4.


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 is an RNA-binding protein.(A) In vitro translated [35S] methionine labelled full-length FMR2, full-length FMRP, N-ter and C-ter proteins were incubated with each RNA homopolymer linked to agarose in the presence of 0.25 M KCl. The same volume (10 μl) of each eluate was analysed by SDS–PAGE followed by fluorography. (B) Labelled N19 probe was incubated in the presence of increasing amounts of recombinant N-ter (lanes 2–4: 0.2, 0.4 and 0.6 pmol, respectively) and C-ter (lanes 5–7: 0.2, 0.4 and 0.6 pmol, respectively) proteins. As a control, the labelled N19 probe was shown in lane 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651778&req=5

Figure 4: FMR2 is an RNA-binding protein.(A) In vitro translated [35S] methionine labelled full-length FMR2, full-length FMRP, N-ter and C-ter proteins were incubated with each RNA homopolymer linked to agarose in the presence of 0.25 M KCl. The same volume (10 μl) of each eluate was analysed by SDS–PAGE followed by fluorography. (B) Labelled N19 probe was incubated in the presence of increasing amounts of recombinant N-ter (lanes 2–4: 0.2, 0.4 and 0.6 pmol, respectively) and C-ter (lanes 5–7: 0.2, 0.4 and 0.6 pmol, respectively) proteins. As a control, the labelled N19 probe was shown in lane 1.
Mentions: To validate our hypothesis, we tested the ability of FMR2 to bind synthetic RNA homopolymers. Indeed, FMR2 selectively binds to poly (G), but not poly (A), poly (C) or poly (U) (Figure 4A) while the control FMRP (Fragile X Mental Retardation Protein), as expected, is able to bind to poly (G) and poly (U) (20). The binding activity to poly (G) was retained by the FMR2 C-ter domain, the same region directing its localization in nuclear speckles (Figure 2), but not by the N-ter domain (Figure 2). Even if no homology with other RNA-binding domains is present in FMR2, two putative RNA-binding motifs were identified within this region (the first between residues 787 and 815 and the second between residues 890 and 917) using the RNABindR program (27). Since FMR2 has affinity for poly (G) RNA, we questioned whether it can bind any RNA containing a stretch of G nucleotides or if it is able to recognize and bind a specific G-rich RNA structure. For this reason, we tested the ability of N-ter and C-ter recombinant FMR2 proteins to bind a RNA probe containing a G-quartet forming structure, the N-19 RNA in a gel retardation assay. Previously, we have shown that FMR1 mRNA contains a G-quartet forming structure, the N19 sequence, localized between nucleotides 1470 and 1896 of FMR1, which is bound by FMRP itself (18,28). Indeed, as for the poly G-sequence increasing amounts of C-ter bind the N19 probe while of N-ter does not interact with the RNA (Figure 4B), suggesting a specificity of FMR2 for G-quartet structure via its C-terminal domain.Figure 4.

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