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BC1-FMRP interaction is modulated by 2'-O-methylation: RNA-binding activity of the tudor domain and translational regulation at synapses.

Lacoux C, Di Marino D, Boyl PP, Zalfa F, Yan B, Ciotti MT, Falconi M, Urlaub H, Achsel T, Mougin A, Caizergues-Ferrer M, Bagni C - Nucleic Acids Res. (2012)

Bottom Line: The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control.These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets.We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine and Biochemical Sciences, Faculty of Medicine, University of Rome Tor Vergata, Via Montpellier, 1. 00133, Rome, Italy.

ABSTRACT
The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control. One component of BC1-containing RNP complexes is the fragile X mental retardation protein (FMRP) that is implicated in translational repression. Peptide mapping and computational simulations show that the tudor domain of FMRP makes specific contacts to BC1 RNA. Endogenous BC1 RNA is 2'-O-methylated in nucleotides that contact the FMRP interface, and methylation can affect this interaction. In the cell body BC1 2'-O-methylations are present in both the nucleus and the cytoplasm, but they are virtually absent at synapses where the FMRP-BC1-mRNA complex exerts its function. These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets. We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP. All together these findings provide further insights into the translational regulation by the FMRP-BC1 complex at synapses.

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BC1 RNA represses mRNA translation at synapses. (A) Cytoplasmic brain extracts from WT and BC1 KO mice were centrifuged through a 15–50% sucrose gradient; absorbance at 254 nm was monitored continuously and plotted against the fraction numbers. (B) Fractions 1–5, corresponding to polysomes (P) and the fractions 6–10 containing mRNPs (mRNP) were pooled and further analysed by RT–qPCR. (C) Translational efficiency of Arc, αCaMKII and Map1B mRNAs from WT (grey histograms) and BC1 KO (black histograms) mice were quantified and expressed as PMP in the histograms. (D) Same as in panel (C) using synaptosomal preparation. Error bars represent SE: *P < 0.05 or **P < 0.01 for BC1 KO versus WT by Student's test, n = 4. (E) Protein levels of Arc, αCaMKII and Map1B from WT (in white) and BC1 KO (in black) from total brain. (F) Same as in (E) using synaptic protein extracts. Error bars represent SE: *P < 0.05 for KO versus WT by Student's test, n = 3.
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gkr1254-F1: BC1 RNA represses mRNA translation at synapses. (A) Cytoplasmic brain extracts from WT and BC1 KO mice were centrifuged through a 15–50% sucrose gradient; absorbance at 254 nm was monitored continuously and plotted against the fraction numbers. (B) Fractions 1–5, corresponding to polysomes (P) and the fractions 6–10 containing mRNPs (mRNP) were pooled and further analysed by RT–qPCR. (C) Translational efficiency of Arc, αCaMKII and Map1B mRNAs from WT (grey histograms) and BC1 KO (black histograms) mice were quantified and expressed as PMP in the histograms. (D) Same as in panel (C) using synaptosomal preparation. Error bars represent SE: *P < 0.05 or **P < 0.01 for BC1 KO versus WT by Student's test, n = 4. (E) Protein levels of Arc, αCaMKII and Map1B from WT (in white) and BC1 KO (in black) from total brain. (F) Same as in (E) using synaptic protein extracts. Error bars represent SE: *P < 0.05 for KO versus WT by Student's test, n = 3.

Mentions: BC1 RNA is highly localized in dendrites and axons and it is thought to regulate local protein synthesis (5,36). Previous reports have shown a functional interaction between BC1 RNA and FMRP at synapses (5,9,37,38). To investigate the contribution of BC1 RNA to FMRP-mediated translational regulation, we performed polysome-mRNP gradient analysis. Cytoplasmic whole-brain extracts from 3 weeks old WT and BC1 KO mice (39) were fractionated on continuous sucrose gradients. Figure 1A shows the sedimentation profiles for the two genotypes. Fractions that contain actively translating polysomes (P) and translationally silent mRNPs (mRNPs), respectively, were pooled and the extracted RNAs were used to determine the translational efficiency by RT–qPCR (as percentage of mRNAs on polysomes, PMP, Figure 1B) of Arc, αCamKII and Map1B mRNAs that are translationally controlled by FMRP (5,40–44). When the polysomal-mRNP analysis was carried out from total brain, no increase in the PMP value was observed for Arc, αCamKII and Map1B mRNAs (Figure 1C). These data show that in total brain of BC1 KO animals, the translational efficiency of some FMRP target mRNAs is not altered compared to WT (Figure 1C).Figure 1.


BC1-FMRP interaction is modulated by 2'-O-methylation: RNA-binding activity of the tudor domain and translational regulation at synapses.

Lacoux C, Di Marino D, Boyl PP, Zalfa F, Yan B, Ciotti MT, Falconi M, Urlaub H, Achsel T, Mougin A, Caizergues-Ferrer M, Bagni C - Nucleic Acids Res. (2012)

BC1 RNA represses mRNA translation at synapses. (A) Cytoplasmic brain extracts from WT and BC1 KO mice were centrifuged through a 15–50% sucrose gradient; absorbance at 254 nm was monitored continuously and plotted against the fraction numbers. (B) Fractions 1–5, corresponding to polysomes (P) and the fractions 6–10 containing mRNPs (mRNP) were pooled and further analysed by RT–qPCR. (C) Translational efficiency of Arc, αCaMKII and Map1B mRNAs from WT (grey histograms) and BC1 KO (black histograms) mice were quantified and expressed as PMP in the histograms. (D) Same as in panel (C) using synaptosomal preparation. Error bars represent SE: *P < 0.05 or **P < 0.01 for BC1 KO versus WT by Student's test, n = 4. (E) Protein levels of Arc, αCaMKII and Map1B from WT (in white) and BC1 KO (in black) from total brain. (F) Same as in (E) using synaptic protein extracts. Error bars represent SE: *P < 0.05 for KO versus WT by Student's test, n = 3.
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Related In: Results  -  Collection

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gkr1254-F1: BC1 RNA represses mRNA translation at synapses. (A) Cytoplasmic brain extracts from WT and BC1 KO mice were centrifuged through a 15–50% sucrose gradient; absorbance at 254 nm was monitored continuously and plotted against the fraction numbers. (B) Fractions 1–5, corresponding to polysomes (P) and the fractions 6–10 containing mRNPs (mRNP) were pooled and further analysed by RT–qPCR. (C) Translational efficiency of Arc, αCaMKII and Map1B mRNAs from WT (grey histograms) and BC1 KO (black histograms) mice were quantified and expressed as PMP in the histograms. (D) Same as in panel (C) using synaptosomal preparation. Error bars represent SE: *P < 0.05 or **P < 0.01 for BC1 KO versus WT by Student's test, n = 4. (E) Protein levels of Arc, αCaMKII and Map1B from WT (in white) and BC1 KO (in black) from total brain. (F) Same as in (E) using synaptic protein extracts. Error bars represent SE: *P < 0.05 for KO versus WT by Student's test, n = 3.
Mentions: BC1 RNA is highly localized in dendrites and axons and it is thought to regulate local protein synthesis (5,36). Previous reports have shown a functional interaction between BC1 RNA and FMRP at synapses (5,9,37,38). To investigate the contribution of BC1 RNA to FMRP-mediated translational regulation, we performed polysome-mRNP gradient analysis. Cytoplasmic whole-brain extracts from 3 weeks old WT and BC1 KO mice (39) were fractionated on continuous sucrose gradients. Figure 1A shows the sedimentation profiles for the two genotypes. Fractions that contain actively translating polysomes (P) and translationally silent mRNPs (mRNPs), respectively, were pooled and the extracted RNAs were used to determine the translational efficiency by RT–qPCR (as percentage of mRNAs on polysomes, PMP, Figure 1B) of Arc, αCamKII and Map1B mRNAs that are translationally controlled by FMRP (5,40–44). When the polysomal-mRNP analysis was carried out from total brain, no increase in the PMP value was observed for Arc, αCamKII and Map1B mRNAs (Figure 1C). These data show that in total brain of BC1 KO animals, the translational efficiency of some FMRP target mRNAs is not altered compared to WT (Figure 1C).Figure 1.

Bottom Line: The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control.These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets.We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine and Biochemical Sciences, Faculty of Medicine, University of Rome Tor Vergata, Via Montpellier, 1. 00133, Rome, Italy.

ABSTRACT
The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control. One component of BC1-containing RNP complexes is the fragile X mental retardation protein (FMRP) that is implicated in translational repression. Peptide mapping and computational simulations show that the tudor domain of FMRP makes specific contacts to BC1 RNA. Endogenous BC1 RNA is 2'-O-methylated in nucleotides that contact the FMRP interface, and methylation can affect this interaction. In the cell body BC1 2'-O-methylations are present in both the nucleus and the cytoplasm, but they are virtually absent at synapses where the FMRP-BC1-mRNA complex exerts its function. These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets. We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP. All together these findings provide further insights into the translational regulation by the FMRP-BC1 complex at synapses.

Show MeSH
Related in: MedlinePlus