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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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SoSLIP Acts as an FMRP-Independent IRES-like Element(A) Diagram of different constructs containing both DsRed and eGFP. These plasmids were modified by insertion of either a linker sequence (pPRIG-empty), the SoSLIP sequence (SoSLI-PRIG), or a characterized IRES (pPRIG-HA-red).(B) Histogram showing eGFP intensity (green) in a FACScan analysis on HeLa cells transfected with pPRIGempty, SoSLI-PRIG, or pPRIG-HA-red vectors. Two-hundred thousand cells positive for DsRed expression were analyzed for each transfection, and three independent experiments were quantified. The mean intensity of eGFP was calculated by the instrument software. Statistical analysis shows a significant difference between the mean intensity of GFP obtained by the pPRIGempty vector and that obtained by the SoSLI-PRIG vector (Student's t-test, **p < 0.01).(C) The same analysis described in (B) was repeated in STEK cells expressing or not expressing the FMR1 transgene. Statistical analysis does not show a significant difference between the mean intensity of GFP in cells expressing or not expressing FMRP. Results are presented as the mean ± SEM.(D) In vitro translated capped and noncapped mRNA luciferase (Luc vector) in WGE. The relative intensity of each band was evaluated by densitometric analysis, and the values obtained are represented in the histograms. Four different experiments were quantified, and results are presented as the mean ± SEM (Student's t-test, ***p < 0.001).(E) The same experiment described in (D) was repeated for the in vitro translation of SoSLIP-Luc mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.(F) The same experiment described in (D) was repeated for the in vitro translation of Sod1 mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.As in (D), in (E) and (F), results are presented as the mean ± SEM.
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pbio-1000016-g008: SoSLIP Acts as an FMRP-Independent IRES-like Element(A) Diagram of different constructs containing both DsRed and eGFP. These plasmids were modified by insertion of either a linker sequence (pPRIG-empty), the SoSLIP sequence (SoSLI-PRIG), or a characterized IRES (pPRIG-HA-red).(B) Histogram showing eGFP intensity (green) in a FACScan analysis on HeLa cells transfected with pPRIGempty, SoSLI-PRIG, or pPRIG-HA-red vectors. Two-hundred thousand cells positive for DsRed expression were analyzed for each transfection, and three independent experiments were quantified. The mean intensity of eGFP was calculated by the instrument software. Statistical analysis shows a significant difference between the mean intensity of GFP obtained by the pPRIGempty vector and that obtained by the SoSLI-PRIG vector (Student's t-test, **p < 0.01).(C) The same analysis described in (B) was repeated in STEK cells expressing or not expressing the FMR1 transgene. Statistical analysis does not show a significant difference between the mean intensity of GFP in cells expressing or not expressing FMRP. Results are presented as the mean ± SEM.(D) In vitro translated capped and noncapped mRNA luciferase (Luc vector) in WGE. The relative intensity of each band was evaluated by densitometric analysis, and the values obtained are represented in the histograms. Four different experiments were quantified, and results are presented as the mean ± SEM (Student's t-test, ***p < 0.001).(E) The same experiment described in (D) was repeated for the in vitro translation of SoSLIP-Luc mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.(F) The same experiment described in (D) was repeated for the in vitro translation of Sod1 mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.As in (D), in (E) and (F), results are presented as the mean ± SEM.

Mentions: Due to its effects on translation, we asked then whether SoSLIP may act as an IRES. For this purpose, we used the pPRIG-HA-red bicistronic vector, where Discosoma sp. red fluorescent protein (DsRed) and enhanced green fluorescent protein (eGFP) are under the control of the same promoter. The DsRed is translated in a cap-dependent manner, whereas eGFP is translated only if an IRES sequence is cloned in front of it, as described [22]. We removed the IRES sequence (pPRIGempty), and we cloned the SoSLIP sequence between the DsRed and the eGFP cDNAs (SoSLI-PRIG) (Figure 8A). After 48 h of transfection, HeLa cells were analyzed by fluorescent-activated cell sorting (FACS), and the eGFP intensity was quantified for 200,000 cells expressing DsRed at a constant intensity. As shown in Figure 8B, we did not observe any green fluorescence in cells transfected with pPRIGempty, confirming that eGFP is not expressed in the absence of an upstream IRES sequence. Conversely, when SoSLIP is placed upstream of eGFP cDNA, green fluorescence becomes readily detectable. However, in this case, the mean eGFP fluorescence intensity was 5.4-fold less than that when using the strong viral IRES in pPRIG-HA (250 and 1,400 arbitrary units (AUs), respectively). The same result was observed following transfection of the neuroblastoma NG108 cell line, neurons, COS cells, and STEK cells expressing FMR1 (unpublished data). No differences were observed in the intensity level of GFP fluorescence from the SoSLI-PRIG vector in cells expressing or not expressing FMRP, suggesting that this protein is not required for the mild IRES-like activity of SoSLIP (Figure 8C). Even if FMRP cannot be considered as an IRES translational activating factor, the observation that SoSLIP may act as an IRES-like sequence is important to understand the role of SoSLIP in translational control. To confirm this result, we produced mRNAs encoding luciferase and SoSLIP-Luc carrying or not carrying the cap modification, and we translated in vitro equal amounts of each mRNA in rabbit reticulocyte lysate (RRL) and in wheat germ extract (WGE). As expected, luciferase mRNA is translated with higher efficiency when the mRNA is capped (Figure 8D). SoSLIP-Luc is translated in a cap-independent manner with efficiency comparable to that obtained in the cap-dependent manner (Figure 8E). The same result was obtained for the translation of Sod1 mRNA in the capped and not capped versions (Figure 8F). No differences were observed using either the RRL or the WGE systems.


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)

SoSLIP Acts as an FMRP-Independent IRES-like Element(A) Diagram of different constructs containing both DsRed and eGFP. These plasmids were modified by insertion of either a linker sequence (pPRIG-empty), the SoSLIP sequence (SoSLI-PRIG), or a characterized IRES (pPRIG-HA-red).(B) Histogram showing eGFP intensity (green) in a FACScan analysis on HeLa cells transfected with pPRIGempty, SoSLI-PRIG, or pPRIG-HA-red vectors. Two-hundred thousand cells positive for DsRed expression were analyzed for each transfection, and three independent experiments were quantified. The mean intensity of eGFP was calculated by the instrument software. Statistical analysis shows a significant difference between the mean intensity of GFP obtained by the pPRIGempty vector and that obtained by the SoSLI-PRIG vector (Student's t-test, **p < 0.01).(C) The same analysis described in (B) was repeated in STEK cells expressing or not expressing the FMR1 transgene. Statistical analysis does not show a significant difference between the mean intensity of GFP in cells expressing or not expressing FMRP. Results are presented as the mean ± SEM.(D) In vitro translated capped and noncapped mRNA luciferase (Luc vector) in WGE. The relative intensity of each band was evaluated by densitometric analysis, and the values obtained are represented in the histograms. Four different experiments were quantified, and results are presented as the mean ± SEM (Student's t-test, ***p < 0.001).(E) The same experiment described in (D) was repeated for the in vitro translation of SoSLIP-Luc mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.(F) The same experiment described in (D) was repeated for the in vitro translation of Sod1 mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.As in (D), in (E) and (F), results are presented as the mean ± SEM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628407&req=5

pbio-1000016-g008: SoSLIP Acts as an FMRP-Independent IRES-like Element(A) Diagram of different constructs containing both DsRed and eGFP. These plasmids were modified by insertion of either a linker sequence (pPRIG-empty), the SoSLIP sequence (SoSLI-PRIG), or a characterized IRES (pPRIG-HA-red).(B) Histogram showing eGFP intensity (green) in a FACScan analysis on HeLa cells transfected with pPRIGempty, SoSLI-PRIG, or pPRIG-HA-red vectors. Two-hundred thousand cells positive for DsRed expression were analyzed for each transfection, and three independent experiments were quantified. The mean intensity of eGFP was calculated by the instrument software. Statistical analysis shows a significant difference between the mean intensity of GFP obtained by the pPRIGempty vector and that obtained by the SoSLI-PRIG vector (Student's t-test, **p < 0.01).(C) The same analysis described in (B) was repeated in STEK cells expressing or not expressing the FMR1 transgene. Statistical analysis does not show a significant difference between the mean intensity of GFP in cells expressing or not expressing FMRP. Results are presented as the mean ± SEM.(D) In vitro translated capped and noncapped mRNA luciferase (Luc vector) in WGE. The relative intensity of each band was evaluated by densitometric analysis, and the values obtained are represented in the histograms. Four different experiments were quantified, and results are presented as the mean ± SEM (Student's t-test, ***p < 0.001).(E) The same experiment described in (D) was repeated for the in vitro translation of SoSLIP-Luc mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.(F) The same experiment described in (D) was repeated for the in vitro translation of Sod1 mRNA. Four different experiments were evaluated, and no statistically significant differences were observed.As in (D), in (E) and (F), results are presented as the mean ± SEM.
Mentions: Due to its effects on translation, we asked then whether SoSLIP may act as an IRES. For this purpose, we used the pPRIG-HA-red bicistronic vector, where Discosoma sp. red fluorescent protein (DsRed) and enhanced green fluorescent protein (eGFP) are under the control of the same promoter. The DsRed is translated in a cap-dependent manner, whereas eGFP is translated only if an IRES sequence is cloned in front of it, as described [22]. We removed the IRES sequence (pPRIGempty), and we cloned the SoSLIP sequence between the DsRed and the eGFP cDNAs (SoSLI-PRIG) (Figure 8A). After 48 h of transfection, HeLa cells were analyzed by fluorescent-activated cell sorting (FACS), and the eGFP intensity was quantified for 200,000 cells expressing DsRed at a constant intensity. As shown in Figure 8B, we did not observe any green fluorescence in cells transfected with pPRIGempty, confirming that eGFP is not expressed in the absence of an upstream IRES sequence. Conversely, when SoSLIP is placed upstream of eGFP cDNA, green fluorescence becomes readily detectable. However, in this case, the mean eGFP fluorescence intensity was 5.4-fold less than that when using the strong viral IRES in pPRIG-HA (250 and 1,400 arbitrary units (AUs), respectively). The same result was observed following transfection of the neuroblastoma NG108 cell line, neurons, COS cells, and STEK cells expressing FMR1 (unpublished data). No differences were observed in the intensity level of GFP fluorescence from the SoSLI-PRIG vector in cells expressing or not expressing FMRP, suggesting that this protein is not required for the mild IRES-like activity of SoSLIP (Figure 8C). Even if FMRP cannot be considered as an IRES translational activating factor, the observation that SoSLIP may act as an IRES-like sequence is important to understand the role of SoSLIP in translational control. To confirm this result, we produced mRNAs encoding luciferase and SoSLIP-Luc carrying or not carrying the cap modification, and we translated in vitro equal amounts of each mRNA in rabbit reticulocyte lysate (RRL) and in wheat germ extract (WGE). As expected, luciferase mRNA is translated with higher efficiency when the mRNA is capped (Figure 8D). SoSLIP-Luc is translated in a cap-independent manner with efficiency comparable to that obtained in the cap-dependent manner (Figure 8E). The same result was obtained for the translation of Sod1 mRNA in the capped and not capped versions (Figure 8F). No differences were observed using either the RRL or the WGE systems.

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.

Show MeSH
Related in: MedlinePlus