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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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Blocking of transcription and splicing affects nuclear speckles localization of FMR2. (A) Blocking of transcription in NG108 neuroblastoma cells by ActD. The treatment affects the morphology and number of nuclear speckles, as revealed by detection of SC35 with anti-SC35 antibody. FMR2 is co-localized with SC35 in control cells and after treatment, as detected by the anti-FMR2 antibody. In ActD-treated cells, FMR2 is also localized in the cytoplasm. The 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analysed, showing a comparable results. (B) Blocking of splicing in SK-N-SH neuroblastoma cells. In U6 antisense-microinjected cells, SC35 is accumulated in enlarged nuclear speckles, as detected by monoclonal anti-SC35 antibody. In these cells, FMR2 is co-localized with SC35 and is also concentrated in the nucleoli, as detected by polyclonal anti-FMR2 antibody. Upper panels, 20× magnification, scale bar 10 μm; lower panels, 63× magnification, scale bar 5 μm. Ten 20× fields were analysed, showing a comparable result.
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Figure 3: Blocking of transcription and splicing affects nuclear speckles localization of FMR2. (A) Blocking of transcription in NG108 neuroblastoma cells by ActD. The treatment affects the morphology and number of nuclear speckles, as revealed by detection of SC35 with anti-SC35 antibody. FMR2 is co-localized with SC35 in control cells and after treatment, as detected by the anti-FMR2 antibody. In ActD-treated cells, FMR2 is also localized in the cytoplasm. The 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analysed, showing a comparable results. (B) Blocking of splicing in SK-N-SH neuroblastoma cells. In U6 antisense-microinjected cells, SC35 is accumulated in enlarged nuclear speckles, as detected by monoclonal anti-SC35 antibody. In these cells, FMR2 is co-localized with SC35 and is also concentrated in the nucleoli, as detected by polyclonal anti-FMR2 antibody. Upper panels, 20× magnification, scale bar 10 μm; lower panels, 63× magnification, scale bar 5 μm. Ten 20× fields were analysed, showing a comparable result.

Mentions: Nuclear speckles are dynamic structures whose size and shape vary according to the level of RNA polymerase II-dependent transcription and splicing activity (25). For this reason, we investigated whether inhibition of splicing or transcription have an impact on FMR2 intracellular localization (21,26). First, we studied the subcellular distribution of SC35 and FMR2 in NG108 cells after blocking transcription by ActD. As expected, SC35 appears redistributed into enlarged, rounded speckles (Figure 3A), similar to other splicing factors (26). Interestingly, we observed that a portion of FMR2 protein is co-localized with SC35. We also blocked splicing in living cells by injecting the antisense U6 oligonucleotide in the SK-N-SH neuroblastoma cell line. This led to the accumulation of SC35 in enlarged speckle domains (Figure 3B). In these conditions FMR2 is partially co-localized with SC35 in these speckles-like structures and also concentrated in nucleoli (Figure 3B). Taken together, these data strongly suggest that FMR2 is involved in splicing and may have RNA-binding properties.Figure 3.


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)

Blocking of transcription and splicing affects nuclear speckles localization of FMR2. (A) Blocking of transcription in NG108 neuroblastoma cells by ActD. The treatment affects the morphology and number of nuclear speckles, as revealed by detection of SC35 with anti-SC35 antibody. FMR2 is co-localized with SC35 in control cells and after treatment, as detected by the anti-FMR2 antibody. In ActD-treated cells, FMR2 is also localized in the cytoplasm. The 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analysed, showing a comparable results. (B) Blocking of splicing in SK-N-SH neuroblastoma cells. In U6 antisense-microinjected cells, SC35 is accumulated in enlarged nuclear speckles, as detected by monoclonal anti-SC35 antibody. In these cells, FMR2 is co-localized with SC35 and is also concentrated in the nucleoli, as detected by polyclonal anti-FMR2 antibody. Upper panels, 20× magnification, scale bar 10 μm; lower panels, 63× magnification, scale bar 5 μm. Ten 20× fields were analysed, showing a comparable result.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Blocking of transcription and splicing affects nuclear speckles localization of FMR2. (A) Blocking of transcription in NG108 neuroblastoma cells by ActD. The treatment affects the morphology and number of nuclear speckles, as revealed by detection of SC35 with anti-SC35 antibody. FMR2 is co-localized with SC35 in control cells and after treatment, as detected by the anti-FMR2 antibody. In ActD-treated cells, FMR2 is also localized in the cytoplasm. The 40× magnification, scale bar 10 μm. Twenty-five 40× fields were analysed, showing a comparable results. (B) Blocking of splicing in SK-N-SH neuroblastoma cells. In U6 antisense-microinjected cells, SC35 is accumulated in enlarged nuclear speckles, as detected by monoclonal anti-SC35 antibody. In these cells, FMR2 is co-localized with SC35 and is also concentrated in the nucleoli, as detected by polyclonal anti-FMR2 antibody. Upper panels, 20× magnification, scale bar 10 μm; lower panels, 63× magnification, scale bar 5 μm. Ten 20× fields were analysed, showing a comparable result.
Mentions: Nuclear speckles are dynamic structures whose size and shape vary according to the level of RNA polymerase II-dependent transcription and splicing activity (25). For this reason, we investigated whether inhibition of splicing or transcription have an impact on FMR2 intracellular localization (21,26). First, we studied the subcellular distribution of SC35 and FMR2 in NG108 cells after blocking transcription by ActD. As expected, SC35 appears redistributed into enlarged, rounded speckles (Figure 3A), similar to other splicing factors (26). Interestingly, we observed that a portion of FMR2 protein is co-localized with SC35. We also blocked splicing in living cells by injecting the antisense U6 oligonucleotide in the SK-N-SH neuroblastoma cell line. This led to the accumulation of SC35 in enlarged speckle domains (Figure 3B). In these conditions FMR2 is partially co-localized with SC35 in these speckles-like structures and also concentrated in nucleoli (Figure 3B). Taken together, these data strongly suggest that FMR2 is involved in splicing and may have RNA-binding properties.Figure 3.

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