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A novel function for fragile X mental retardation protein in translational activation.

Bechara EG, Didiot MC, Melko M, Davidovic L, Bensaid M, Martin P, Castets M, Pognonec P, Khandjian EW, Moine H, Bardoni B - PLoS Biol. (2009)

Bottom Line: To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the "kissing complex," which both induce translational repression in the presence of FMRP.The absence of FMRP results in decreased expression of Sod1.Because it has been observed that brain metabolism of FMR1 mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the "kissing complex," which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

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Impact of SoSLIP on Translational Regulation(A) Effect of SoSLIP sequence upon luciferase expression: luciferase activities of Luc or SoSLIP-Luc vectors in primary neurons and STEK cells. Three independent experiments with three replicates, done in triplicate, for each transfection were quantified. For each transfection, firefly(F) luciferase (luc) activity was normalized by Renilla (R) luciferase (luc) activity. Results are presented as the mean ± SEM (Student's t-test, **p < 0.01).(B) Activity of SoSLIP-Luc in neurons and STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized by Rluc activity. Results presented here represent the mean ± SEM of the ratio of SoSLIP-Luc to Luc activities (Student's t-test, **p < 0.01).(C) Schematic representation of the wild-type SoSLIP sequence and its three mutants (SL1, SL2, and SL3).(D) Binding affinity of FMRP to wild-type SoSLIP and SL1, SL2, and SL3 mutants. Filter binding assay using radiolabeled SoSLIP and unlabeled cold RNA competitors SoSLIP, Sod1–3′ region, SL1, SL2, and SL3. All of the results obtained in the filter binding assay are listed in Table S2.(E) Effect of SoSLIP mutants (SL1-Luc, SL2-Luc, and SL3-Luc) on luciferase expression in STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized to Rluc activity. Results presented here represent the mean of the ratio of SoSLIP-Luc to Luc, SL1-Luc to Luc, SL2-Luc to Luc, and SL3-Luc to Luc. The luciferase activities of the three mutants were compared to wild-type SoSLIP luciferase activity in cells expressing FMRP, and the difference was significant in all cases (Student's t-test, **p < 0.01). The same analysis was repeated in cells not expressing FMRP, and the difference was significant in all cases (Student's t-test, ##p < 0.01). The luciferase activity of each mutant in cells expressing or not expressing FMRP was evaluated. For mutants SL2 and SL3, the reduction of lucferase activity observed in Fmr1  cells was statistically significant. These results are presented as the mean ± SEM (Student's t-test, °°p < 0.01). For mutant SL1, no significant reduction of luciferase activity was observed in cells not expressing FMRP compared with cells expressing FMRP. These results are presented as the mean ± SEM. RLU, relative luciferase units.
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pbio-1000016-g007: Impact of SoSLIP on Translational Regulation(A) Effect of SoSLIP sequence upon luciferase expression: luciferase activities of Luc or SoSLIP-Luc vectors in primary neurons and STEK cells. Three independent experiments with three replicates, done in triplicate, for each transfection were quantified. For each transfection, firefly(F) luciferase (luc) activity was normalized by Renilla (R) luciferase (luc) activity. Results are presented as the mean ± SEM (Student's t-test, **p < 0.01).(B) Activity of SoSLIP-Luc in neurons and STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized by Rluc activity. Results presented here represent the mean ± SEM of the ratio of SoSLIP-Luc to Luc activities (Student's t-test, **p < 0.01).(C) Schematic representation of the wild-type SoSLIP sequence and its three mutants (SL1, SL2, and SL3).(D) Binding affinity of FMRP to wild-type SoSLIP and SL1, SL2, and SL3 mutants. Filter binding assay using radiolabeled SoSLIP and unlabeled cold RNA competitors SoSLIP, Sod1–3′ region, SL1, SL2, and SL3. All of the results obtained in the filter binding assay are listed in Table S2.(E) Effect of SoSLIP mutants (SL1-Luc, SL2-Luc, and SL3-Luc) on luciferase expression in STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized to Rluc activity. Results presented here represent the mean of the ratio of SoSLIP-Luc to Luc, SL1-Luc to Luc, SL2-Luc to Luc, and SL3-Luc to Luc. The luciferase activities of the three mutants were compared to wild-type SoSLIP luciferase activity in cells expressing FMRP, and the difference was significant in all cases (Student's t-test, **p < 0.01). The same analysis was repeated in cells not expressing FMRP, and the difference was significant in all cases (Student's t-test, ##p < 0.01). The luciferase activity of each mutant in cells expressing or not expressing FMRP was evaluated. For mutants SL2 and SL3, the reduction of lucferase activity observed in Fmr1 cells was statistically significant. These results are presented as the mean ± SEM (Student's t-test, °°p < 0.01). For mutant SL1, no significant reduction of luciferase activity was observed in cells not expressing FMRP compared with cells expressing FMRP. These results are presented as the mean ± SEM. RLU, relative luciferase units.

Mentions: To confirm the positive role of FMRP in translational modulation of Sod1 expression by the interaction with the SoSLIP RNA structure, we cloned this sequence upstream of the luciferase gene in the pcDNA3.1 zeo vector (Luc) to evaluate the effect of the presence of SoSLIP on the expression of a reporter protein. We transfected primary cultured hippocampal neurons with the SoSLIP-luciferase vector (SoSLIP-Luc) or with the Luc vector, and we tested luciferase activity, showing on average an 8-fold increase when SoSLIP is placed upstream of the reporter (Figure 7A). A similar result was obtained in FMR1-expressing STEK cells (Figure 7A). Analysis of luciferase mRNA levels tested by qRT-PCR revealed that the presence of SoSLIP did not affect the mRNA expression level or stability of the dowstream reporter gene (Figure S4A and S4B). These results indicate that SoSLIP behaves per se as a translational activator in both cell types. We then transfected the same plasmid in primary hippocampal neurons obtained from normal and Fmr1 mice and in STEK cells expressing or not expressing the FMR1 transgene. Indeed, in the absence of FMRP, luciferase activity resulted in a 2-fold reduction as compared with that in the presence of FMRP (wild-type condition) (Figure 7B). These data suggest that the presence of FMRP potentiates the ability of the SoSLIP sequence to positively modulate the expression of a downstream coding sequence independently of the cellular type. In addition, our findings are compatible with the notion that FMRP's roles in translation might be positive or negative as discussed [1,13]. To test the functional importance of SoSLIP stem loops, we disrupted each of the three stem loops by site-directed mutagenesis (Figure 7C). Using a filter binding assay, we then tested the ability of each mutant to compete for the binding of the FMRP/SoSLIP interaction. As shown in Figure 7D, the SL1 mutant is able to fully compete for SoSLIP binding (4 nM cold SL1 probe competes for 50% of wild-type SoSLIP), indicating that the disruption of SL1 does not affect FMRP/SoSLIP interaction. Conversely, the two SL2 and SL3 mutants poorly compete for SoSLIP binding to FMRP (60 nM concentrations of both cold probes compete for 50% of SoSLIP). The disruption of these two stem loops reduces their affinity for FMRP binding (Figure 7D) but did not abolish this binding, as suggested by comparing with the competition of an RNA sequence not bound by FMRP (Figure 7D, 3′ UTR Sod1 RNA as the cold competitor). All three mutations affect the SoSLIP translational enhancer properties, reducing the level of luciferase activity (Figure 7E), if compared with the luciferase activity of the SoSLIP-Luc construct. Indeed, the activities of SL1-Luc, SL2-Luc, and SL3-Luc are reduced by 80%, 50%, and 60%, respectively, if compared with SoSLIP-Luc activity when these constructs have been transfected in cells expressing FMRP. The activities of SL1-Luc, SL2-Luc, and SL3-Luc are also reduced (62%, 44%, and 50%, respectively), if compared with SoSLIP-Luc activity when these constructs have been transfected in cells not expressing FMRP. Furthermore, in Fmr1 knockout cells, the two SL2 and SL3 mutants have a reduced translational enhancing activity if compared with their activity in wild-type cells (Figure 7E) (40% and 33% reduced activity, respectively), confirming the data obtained by the in vitro binding. Surprisingly, the absence of FMRP does not modify the impact of the SL1 mutant on luciferase activity (Figure 7E), suggesting that also the SL1 integrity is necessary for the correct function of FMRP. To confirm that the effect of the mutants was only affecting translation efficiency, the expression and the stability of the mRNAs of all three SoSLIP mutants were tested, and no differences were observed with the wild-type mRNA, in the presence or in the absence of FMRP (Figure S4A–S4C).


A novel function for fragile X mental retardation protein in translational activation.

Bechara EG, Didiot MC, Melko M, Davidovic L, Bensaid M, Martin P, Castets M, Pognonec P, Khandjian EW, Moine H, Bardoni B - PLoS Biol. (2009)

Impact of SoSLIP on Translational Regulation(A) Effect of SoSLIP sequence upon luciferase expression: luciferase activities of Luc or SoSLIP-Luc vectors in primary neurons and STEK cells. Three independent experiments with three replicates, done in triplicate, for each transfection were quantified. For each transfection, firefly(F) luciferase (luc) activity was normalized by Renilla (R) luciferase (luc) activity. Results are presented as the mean ± SEM (Student's t-test, **p < 0.01).(B) Activity of SoSLIP-Luc in neurons and STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized by Rluc activity. Results presented here represent the mean ± SEM of the ratio of SoSLIP-Luc to Luc activities (Student's t-test, **p < 0.01).(C) Schematic representation of the wild-type SoSLIP sequence and its three mutants (SL1, SL2, and SL3).(D) Binding affinity of FMRP to wild-type SoSLIP and SL1, SL2, and SL3 mutants. Filter binding assay using radiolabeled SoSLIP and unlabeled cold RNA competitors SoSLIP, Sod1–3′ region, SL1, SL2, and SL3. All of the results obtained in the filter binding assay are listed in Table S2.(E) Effect of SoSLIP mutants (SL1-Luc, SL2-Luc, and SL3-Luc) on luciferase expression in STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized to Rluc activity. Results presented here represent the mean of the ratio of SoSLIP-Luc to Luc, SL1-Luc to Luc, SL2-Luc to Luc, and SL3-Luc to Luc. The luciferase activities of the three mutants were compared to wild-type SoSLIP luciferase activity in cells expressing FMRP, and the difference was significant in all cases (Student's t-test, **p < 0.01). The same analysis was repeated in cells not expressing FMRP, and the difference was significant in all cases (Student's t-test, ##p < 0.01). The luciferase activity of each mutant in cells expressing or not expressing FMRP was evaluated. For mutants SL2 and SL3, the reduction of lucferase activity observed in Fmr1  cells was statistically significant. These results are presented as the mean ± SEM (Student's t-test, °°p < 0.01). For mutant SL1, no significant reduction of luciferase activity was observed in cells not expressing FMRP compared with cells expressing FMRP. These results are presented as the mean ± SEM. RLU, relative luciferase units.
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pbio-1000016-g007: Impact of SoSLIP on Translational Regulation(A) Effect of SoSLIP sequence upon luciferase expression: luciferase activities of Luc or SoSLIP-Luc vectors in primary neurons and STEK cells. Three independent experiments with three replicates, done in triplicate, for each transfection were quantified. For each transfection, firefly(F) luciferase (luc) activity was normalized by Renilla (R) luciferase (luc) activity. Results are presented as the mean ± SEM (Student's t-test, **p < 0.01).(B) Activity of SoSLIP-Luc in neurons and STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized by Rluc activity. Results presented here represent the mean ± SEM of the ratio of SoSLIP-Luc to Luc activities (Student's t-test, **p < 0.01).(C) Schematic representation of the wild-type SoSLIP sequence and its three mutants (SL1, SL2, and SL3).(D) Binding affinity of FMRP to wild-type SoSLIP and SL1, SL2, and SL3 mutants. Filter binding assay using radiolabeled SoSLIP and unlabeled cold RNA competitors SoSLIP, Sod1–3′ region, SL1, SL2, and SL3. All of the results obtained in the filter binding assay are listed in Table S2.(E) Effect of SoSLIP mutants (SL1-Luc, SL2-Luc, and SL3-Luc) on luciferase expression in STEK cells expressing or not expressing FMRP. Three independent experiments in triplicate for each transfection were quantified. For each transfection, Fluc activity was normalized to Rluc activity. Results presented here represent the mean of the ratio of SoSLIP-Luc to Luc, SL1-Luc to Luc, SL2-Luc to Luc, and SL3-Luc to Luc. The luciferase activities of the three mutants were compared to wild-type SoSLIP luciferase activity in cells expressing FMRP, and the difference was significant in all cases (Student's t-test, **p < 0.01). The same analysis was repeated in cells not expressing FMRP, and the difference was significant in all cases (Student's t-test, ##p < 0.01). The luciferase activity of each mutant in cells expressing or not expressing FMRP was evaluated. For mutants SL2 and SL3, the reduction of lucferase activity observed in Fmr1 cells was statistically significant. These results are presented as the mean ± SEM (Student's t-test, °°p < 0.01). For mutant SL1, no significant reduction of luciferase activity was observed in cells not expressing FMRP compared with cells expressing FMRP. These results are presented as the mean ± SEM. RLU, relative luciferase units.
Mentions: To confirm the positive role of FMRP in translational modulation of Sod1 expression by the interaction with the SoSLIP RNA structure, we cloned this sequence upstream of the luciferase gene in the pcDNA3.1 zeo vector (Luc) to evaluate the effect of the presence of SoSLIP on the expression of a reporter protein. We transfected primary cultured hippocampal neurons with the SoSLIP-luciferase vector (SoSLIP-Luc) or with the Luc vector, and we tested luciferase activity, showing on average an 8-fold increase when SoSLIP is placed upstream of the reporter (Figure 7A). A similar result was obtained in FMR1-expressing STEK cells (Figure 7A). Analysis of luciferase mRNA levels tested by qRT-PCR revealed that the presence of SoSLIP did not affect the mRNA expression level or stability of the dowstream reporter gene (Figure S4A and S4B). These results indicate that SoSLIP behaves per se as a translational activator in both cell types. We then transfected the same plasmid in primary hippocampal neurons obtained from normal and Fmr1 mice and in STEK cells expressing or not expressing the FMR1 transgene. Indeed, in the absence of FMRP, luciferase activity resulted in a 2-fold reduction as compared with that in the presence of FMRP (wild-type condition) (Figure 7B). These data suggest that the presence of FMRP potentiates the ability of the SoSLIP sequence to positively modulate the expression of a downstream coding sequence independently of the cellular type. In addition, our findings are compatible with the notion that FMRP's roles in translation might be positive or negative as discussed [1,13]. To test the functional importance of SoSLIP stem loops, we disrupted each of the three stem loops by site-directed mutagenesis (Figure 7C). Using a filter binding assay, we then tested the ability of each mutant to compete for the binding of the FMRP/SoSLIP interaction. As shown in Figure 7D, the SL1 mutant is able to fully compete for SoSLIP binding (4 nM cold SL1 probe competes for 50% of wild-type SoSLIP), indicating that the disruption of SL1 does not affect FMRP/SoSLIP interaction. Conversely, the two SL2 and SL3 mutants poorly compete for SoSLIP binding to FMRP (60 nM concentrations of both cold probes compete for 50% of SoSLIP). The disruption of these two stem loops reduces their affinity for FMRP binding (Figure 7D) but did not abolish this binding, as suggested by comparing with the competition of an RNA sequence not bound by FMRP (Figure 7D, 3′ UTR Sod1 RNA as the cold competitor). All three mutations affect the SoSLIP translational enhancer properties, reducing the level of luciferase activity (Figure 7E), if compared with the luciferase activity of the SoSLIP-Luc construct. Indeed, the activities of SL1-Luc, SL2-Luc, and SL3-Luc are reduced by 80%, 50%, and 60%, respectively, if compared with SoSLIP-Luc activity when these constructs have been transfected in cells expressing FMRP. The activities of SL1-Luc, SL2-Luc, and SL3-Luc are also reduced (62%, 44%, and 50%, respectively), if compared with SoSLIP-Luc activity when these constructs have been transfected in cells not expressing FMRP. Furthermore, in Fmr1 knockout cells, the two SL2 and SL3 mutants have a reduced translational enhancing activity if compared with their activity in wild-type cells (Figure 7E) (40% and 33% reduced activity, respectively), confirming the data obtained by the in vitro binding. Surprisingly, the absence of FMRP does not modify the impact of the SL1 mutant on luciferase activity (Figure 7E), suggesting that also the SL1 integrity is necessary for the correct function of FMRP. To confirm that the effect of the mutants was only affecting translation efficiency, the expression and the stability of the mRNAs of all three SoSLIP mutants were tested, and no differences were observed with the wild-type mRNA, in the presence or in the absence of FMRP (Figure S4A–S4C).

Bottom Line: To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the "kissing complex," which both induce translational repression in the presence of FMRP.The absence of FMRP results in decreased expression of Sod1.Because it has been observed that brain metabolism of FMR1 mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the "kissing complex," which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

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Related in: MedlinePlus