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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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FMRP Specifically Binds Sod1 mRNAFMRP binding to Sod1 mRNA is not dependent on K+. Labeled G-quartet RNA (N19) or Sod1 full-length mRNA were incubated with increasing amounts of recombinant His-FMRP in the presence of K+(A) or Na+(B). FMRP/Sod1 binding was not affected by ionic conditions, whereas, as expected, the presence of Na+ affected FMRP binding to N19.(C) Gel-shift experiments were performed using a 32P-labeled N19 probe incubated with 0.1 pmol of recombinant His-tagged FMRP in the presence of increasing amounts of unlabeled competitors, ranging from 10−9 to 10−7M [lanes 3–5 (N19), lanes 6–8 (Sod1), lanes 9–11 (N8)]. Lane 1, no protein control; lane 2, no competitor control. Note that both N19 (positive control) and Sod1 compete equally well for binding to FMRP, whereas N8 (negative control) only competes at high concentrations (nonspecific binding). All data obtained in these experiments are listed in Table S2.
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pbio-1000016-g001: FMRP Specifically Binds Sod1 mRNAFMRP binding to Sod1 mRNA is not dependent on K+. Labeled G-quartet RNA (N19) or Sod1 full-length mRNA were incubated with increasing amounts of recombinant His-FMRP in the presence of K+(A) or Na+(B). FMRP/Sod1 binding was not affected by ionic conditions, whereas, as expected, the presence of Na+ affected FMRP binding to N19.(C) Gel-shift experiments were performed using a 32P-labeled N19 probe incubated with 0.1 pmol of recombinant His-tagged FMRP in the presence of increasing amounts of unlabeled competitors, ranging from 10−9 to 10−7M [lanes 3–5 (N19), lanes 6–8 (Sod1), lanes 9–11 (N8)]. Lane 1, no protein control; lane 2, no competitor control. Note that both N19 (positive control) and Sod1 compete equally well for binding to FMRP, whereas N8 (negative control) only competes at high concentrations (nonspecific binding). All data obtained in these experiments are listed in Table S2.

Mentions: With the goal to find novel mRNA structures specifically recognized by FMRP, we performed a systematic analysis of known FMRP mRNA targets, focusing on those that have been shown to interact in vivo with FMRP by the antibody-positioned RNA amplification (APRA) technique [13]. First, we excluded the presence of already known structures bound by FMRP in these mRNA targets by screening their capacity to bind a recombinant FMRP in the presence of Na+, K+, or Mg2+. Indeed, K+ ions stabilize the G-quartet RNA structure, leading to a robust interaction with FMRP [4], whereas Mg2+ favors FMRP/kissing complex RNA interaction [2]. This analysis resulted in the characterization of FMRP/Sod1 interaction, which takes place in the presence of K+ (Figure 1A) and is not affected by the presence of Na+ (Figure 1B), whereas, as expected, Na+ affects the binding of FMRP to the N19 sequence that contains the G-quartet present in FMR1 mRNA (nucleotide (nt) 1470–1496) [4]. Moreover, to definitely exclude the presence of a G-quartet structure in Sod1 mRNA, we performed a reverse transcriptase (RT) elongation reaction assay, as previously described [4]. In the presence of K+, G-quartet RNA is very stable, blocking RT progression at its 3′ edge and resulting in a truncated transcription product. Conversely, in the presence of Na+, G-quartet structures are destabilized, and the RT can proceed to the end of the RNA [4] . The RT elongation test on Sod1 mRNA did not reveal any K+-dependent stop of the polymerase (Figure S1), demonstrating that Sod1 mRNA is not able to form a G-quartet structure. Moreover, FMRP/Sod1 interaction was not dependent on the presence of Mg2+, which is necessary to stabilize the “kissing complex” RNA structure (unpublished data) [2]. Taken together, these findings suggest that FMRP binds to Sod1 mRNA via a novel sequence/structure. We continued the characterization of the FMRP/Sod1 mRNA interaction by testing the ability of Sod1 mRNA to compete for the binding of the FMRP/G-quartet RNA structure [4]. Indeed, 5 nM unlabeled Sod1 mRNA competed very efficiently (65%) with the previously identified N19 FMRP binding site in a gel-shift assay, whereas a negative control, N8 RNA (corresponding to nt 1–654 of FMR1 mRNA that does not contain the G-quartet), was not able to compete for the same interaction (Figure 1C). To precisely define the region of Sod1 mRNA interacting with FMRP, we generated three different constructs from Sod1 encompassing its full-length cDNA: its 5′ UTR and a portion of its coding region (Sod1–5′ region), a central part of the coding region (Sod1-mid region), and a fragment overlapping the end of the coding region and the 3′ UTR (Sod1–3′ region) (Figure 2A). RNA sequences corresponding to each fragment were produced and tested for their ability to interact with FMRP. Only the Sod1–5′ region (spanning nt −70 to +148 of Sod1 mRNA) competed with N19 binding to FMRP with the same affinity as the full-length Sod1 mRNA (3 nM concentrations of both cold probes compete for 50% of FMRP/N19 binding) (Figure 2B). To identify the sequence of Sod1 mRNA that is recognized and bound by FMRP, we employed a site boundary determination method [4]. In this experiment, the 3′- or 5′-end-labeled Sod1–5′ RNA was treated by mild alkaline hydrolysis in order to generate a pool of smaller fragments. The RNA fragments retaining the capacity to bind FMRP were selected on immobilized glutathione-S-transferase (GST)-FMRP, as previously described [4]. Bound RNAs were analyzed by electrophoresis on a denaturing polyacrylamide gel (not shown). The border positions were at −30 and +34 for 3′- and 5′-end-labeled fragments, respectively. This technique allowed us to define a 64-base region spanning both sides of the Sod1 AUG start codon that is protected by FMRP. We subcloned this sequence, and we synthesized its corresponding RNA, generating the Sod1–64 RNA. This RNA was bound specifically by FMRP, because it was able to compete for the FMRP/Sod1 full-length mRNA interaction (Figure 2C). Interestingly, the FMRP/Sod1–64 interaction is competed for by the N19 G-quartet-containing RNA to the same extent (unpublished data).


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)

FMRP Specifically Binds Sod1 mRNAFMRP binding to Sod1 mRNA is not dependent on K+. Labeled G-quartet RNA (N19) or Sod1 full-length mRNA were incubated with increasing amounts of recombinant His-FMRP in the presence of K+(A) or Na+(B). FMRP/Sod1 binding was not affected by ionic conditions, whereas, as expected, the presence of Na+ affected FMRP binding to N19.(C) Gel-shift experiments were performed using a 32P-labeled N19 probe incubated with 0.1 pmol of recombinant His-tagged FMRP in the presence of increasing amounts of unlabeled competitors, ranging from 10−9 to 10−7M [lanes 3–5 (N19), lanes 6–8 (Sod1), lanes 9–11 (N8)]. Lane 1, no protein control; lane 2, no competitor control. Note that both N19 (positive control) and Sod1 compete equally well for binding to FMRP, whereas N8 (negative control) only competes at high concentrations (nonspecific binding). All data obtained in these experiments are listed in Table S2.
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Related In: Results  -  Collection

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

pbio-1000016-g001: FMRP Specifically Binds Sod1 mRNAFMRP binding to Sod1 mRNA is not dependent on K+. Labeled G-quartet RNA (N19) or Sod1 full-length mRNA were incubated with increasing amounts of recombinant His-FMRP in the presence of K+(A) or Na+(B). FMRP/Sod1 binding was not affected by ionic conditions, whereas, as expected, the presence of Na+ affected FMRP binding to N19.(C) Gel-shift experiments were performed using a 32P-labeled N19 probe incubated with 0.1 pmol of recombinant His-tagged FMRP in the presence of increasing amounts of unlabeled competitors, ranging from 10−9 to 10−7M [lanes 3–5 (N19), lanes 6–8 (Sod1), lanes 9–11 (N8)]. Lane 1, no protein control; lane 2, no competitor control. Note that both N19 (positive control) and Sod1 compete equally well for binding to FMRP, whereas N8 (negative control) only competes at high concentrations (nonspecific binding). All data obtained in these experiments are listed in Table S2.
Mentions: With the goal to find novel mRNA structures specifically recognized by FMRP, we performed a systematic analysis of known FMRP mRNA targets, focusing on those that have been shown to interact in vivo with FMRP by the antibody-positioned RNA amplification (APRA) technique [13]. First, we excluded the presence of already known structures bound by FMRP in these mRNA targets by screening their capacity to bind a recombinant FMRP in the presence of Na+, K+, or Mg2+. Indeed, K+ ions stabilize the G-quartet RNA structure, leading to a robust interaction with FMRP [4], whereas Mg2+ favors FMRP/kissing complex RNA interaction [2]. This analysis resulted in the characterization of FMRP/Sod1 interaction, which takes place in the presence of K+ (Figure 1A) and is not affected by the presence of Na+ (Figure 1B), whereas, as expected, Na+ affects the binding of FMRP to the N19 sequence that contains the G-quartet present in FMR1 mRNA (nucleotide (nt) 1470–1496) [4]. Moreover, to definitely exclude the presence of a G-quartet structure in Sod1 mRNA, we performed a reverse transcriptase (RT) elongation reaction assay, as previously described [4]. In the presence of K+, G-quartet RNA is very stable, blocking RT progression at its 3′ edge and resulting in a truncated transcription product. Conversely, in the presence of Na+, G-quartet structures are destabilized, and the RT can proceed to the end of the RNA [4] . The RT elongation test on Sod1 mRNA did not reveal any K+-dependent stop of the polymerase (Figure S1), demonstrating that Sod1 mRNA is not able to form a G-quartet structure. Moreover, FMRP/Sod1 interaction was not dependent on the presence of Mg2+, which is necessary to stabilize the “kissing complex” RNA structure (unpublished data) [2]. Taken together, these findings suggest that FMRP binds to Sod1 mRNA via a novel sequence/structure. We continued the characterization of the FMRP/Sod1 mRNA interaction by testing the ability of Sod1 mRNA to compete for the binding of the FMRP/G-quartet RNA structure [4]. Indeed, 5 nM unlabeled Sod1 mRNA competed very efficiently (65%) with the previously identified N19 FMRP binding site in a gel-shift assay, whereas a negative control, N8 RNA (corresponding to nt 1–654 of FMR1 mRNA that does not contain the G-quartet), was not able to compete for the same interaction (Figure 1C). To precisely define the region of Sod1 mRNA interacting with FMRP, we generated three different constructs from Sod1 encompassing its full-length cDNA: its 5′ UTR and a portion of its coding region (Sod1–5′ region), a central part of the coding region (Sod1-mid region), and a fragment overlapping the end of the coding region and the 3′ UTR (Sod1–3′ region) (Figure 2A). RNA sequences corresponding to each fragment were produced and tested for their ability to interact with FMRP. Only the Sod1–5′ region (spanning nt −70 to +148 of Sod1 mRNA) competed with N19 binding to FMRP with the same affinity as the full-length Sod1 mRNA (3 nM concentrations of both cold probes compete for 50% of FMRP/N19 binding) (Figure 2B). To identify the sequence of Sod1 mRNA that is recognized and bound by FMRP, we employed a site boundary determination method [4]. In this experiment, the 3′- or 5′-end-labeled Sod1–5′ RNA was treated by mild alkaline hydrolysis in order to generate a pool of smaller fragments. The RNA fragments retaining the capacity to bind FMRP were selected on immobilized glutathione-S-transferase (GST)-FMRP, as previously described [4]. Bound RNAs were analyzed by electrophoresis on a denaturing polyacrylamide gel (not shown). The border positions were at −30 and +34 for 3′- and 5′-end-labeled fragments, respectively. This technique allowed us to define a 64-base region spanning both sides of the Sod1 AUG start codon that is protected by FMRP. We subcloned this sequence, and we synthesized its corresponding RNA, generating the Sod1–64 RNA. This RNA was bound specifically by FMRP, because it was able to compete for the FMRP/Sod1 full-length mRNA interaction (Figure 2C). Interestingly, the FMRP/Sod1–64 interaction is competed for by the N19 G-quartet-containing RNA to the same extent (unpublished data).

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