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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 is 2′-O-methylated. (A) Primers used to detect BC1 RNA 2′-O-methylations: BMN297, BMN155 are indicated on the BC1 secondary structure. (B) Low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA from WT mice (lanes 5 and 6), on the BC1 transcript (lanes 11 and 12), and on RNA from synaptoneurosomes (lanes 13 and 14). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration; stops due to low dNTP indicate the presence of a 2′-O-methylation. Sequencing reactions are shown in lanes 1–4 and 7–10. (C) Same as in (B) using the BMN297 primer; sequencing (lanes 1–4), low dNTP concentration primer extension analysis on total brain (lanes 5 and 6), on BC1 transcript (lanes 7 and 8) and on synaptosomal preparation (lanes 9 and 10). (D) Same as in (C) using total brain (lanes 5 and 6), cytoplasmic (lanes 7 and 8) and nuclear extracts (lanes 9 and 10).
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gkr1254-F2: BC1 RNA is 2′-O-methylated. (A) Primers used to detect BC1 RNA 2′-O-methylations: BMN297, BMN155 are indicated on the BC1 secondary structure. (B) Low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA from WT mice (lanes 5 and 6), on the BC1 transcript (lanes 11 and 12), and on RNA from synaptoneurosomes (lanes 13 and 14). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration; stops due to low dNTP indicate the presence of a 2′-O-methylation. Sequencing reactions are shown in lanes 1–4 and 7–10. (C) Same as in (B) using the BMN297 primer; sequencing (lanes 1–4), low dNTP concentration primer extension analysis on total brain (lanes 5 and 6), on BC1 transcript (lanes 7 and 8) and on synaptosomal preparation (lanes 9 and 10). (D) Same as in (C) using total brain (lanes 5 and 6), cytoplasmic (lanes 7 and 8) and nuclear extracts (lanes 9 and 10).

Mentions: We first verified the efficacy of the previously established methodology (26–28) by analysing, in mouse brain, the well-documented U2 snRNA 2′-O-methylations (47) (Supplementary Figure S2A). Using the BC1-specific primers BMN155 and BMN297 (Figure 2A) and total brain RNA, 2′-O-methylations were clearly detected in the 5′-stem-loop at positions G46 (Gm46), C47 (Cm47) and G56 (Gm56) (Figure 2B and C: compare lanes 5 and 6; 2′-O-methylations are detected by strong signals with less dNTPs, n = 5). No 2′-O-methylations were observed using in vitro-transcribed BC1 RNA (Figure 2B and C, lanes 11 and 12 and lanes 7 and 8, respectively), or in the region located between the two hairpins of the endogenous BC1 RNA (nucleotides 75–127, data not shown). Since the FMRP–BC1 complex controls mRNA translation at synapses (5,6) (Figure 1D and F) we prepared synaptoneurosomes in order to analyse the 2′-O-methylation status of BC1 RNA at synapses. In this case, no 2′-O-methylations were detected on the synaptic BC1 RNA (Figure 2B and C, compare lanes 13, 14 and 9 and 10, respectively, n = 4). The presence of intact BC1 RNA molecules was verified by the presence of a stop of elongation at nucleotide 1 of BC1 RNA (Supplementary Figure S2B). Because BC1 RNA was found differentially 2′-O-methylated in the cell body versus the synapses, we investigated its methylation status in the nucleus. Nuclear and cytoplasmic compartments, used for this analysis, were revealed by the presence and enrichment for dyskerin and Glyceraldehyde 3-phosphate dehydrogenase (Supplementary Figure S1C). As shown in Figure 2D, BC1 RNA from the cytoplasmic or nuclear fractions is 2′-O-methylated (compare lanes 7 and 8 and lanes 9 and 10 for cytoplasmic and nuclear fraction, respectively) underlining that absence of these 2′-O-methylations may have a function at synapses. These findings suggest that BC1 2′-O-methylations occur in nucleus.Figure 2.


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 is 2′-O-methylated. (A) Primers used to detect BC1 RNA 2′-O-methylations: BMN297, BMN155 are indicated on the BC1 secondary structure. (B) Low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA from WT mice (lanes 5 and 6), on the BC1 transcript (lanes 11 and 12), and on RNA from synaptoneurosomes (lanes 13 and 14). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration; stops due to low dNTP indicate the presence of a 2′-O-methylation. Sequencing reactions are shown in lanes 1–4 and 7–10. (C) Same as in (B) using the BMN297 primer; sequencing (lanes 1–4), low dNTP concentration primer extension analysis on total brain (lanes 5 and 6), on BC1 transcript (lanes 7 and 8) and on synaptosomal preparation (lanes 9 and 10). (D) Same as in (C) using total brain (lanes 5 and 6), cytoplasmic (lanes 7 and 8) and nuclear extracts (lanes 9 and 10).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351191&req=5

gkr1254-F2: BC1 RNA is 2′-O-methylated. (A) Primers used to detect BC1 RNA 2′-O-methylations: BMN297, BMN155 are indicated on the BC1 secondary structure. (B) Low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA from WT mice (lanes 5 and 6), on the BC1 transcript (lanes 11 and 12), and on RNA from synaptoneurosomes (lanes 13 and 14). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration; stops due to low dNTP indicate the presence of a 2′-O-methylation. Sequencing reactions are shown in lanes 1–4 and 7–10. (C) Same as in (B) using the BMN297 primer; sequencing (lanes 1–4), low dNTP concentration primer extension analysis on total brain (lanes 5 and 6), on BC1 transcript (lanes 7 and 8) and on synaptosomal preparation (lanes 9 and 10). (D) Same as in (C) using total brain (lanes 5 and 6), cytoplasmic (lanes 7 and 8) and nuclear extracts (lanes 9 and 10).
Mentions: We first verified the efficacy of the previously established methodology (26–28) by analysing, in mouse brain, the well-documented U2 snRNA 2′-O-methylations (47) (Supplementary Figure S2A). Using the BC1-specific primers BMN155 and BMN297 (Figure 2A) and total brain RNA, 2′-O-methylations were clearly detected in the 5′-stem-loop at positions G46 (Gm46), C47 (Cm47) and G56 (Gm56) (Figure 2B and C: compare lanes 5 and 6; 2′-O-methylations are detected by strong signals with less dNTPs, n = 5). No 2′-O-methylations were observed using in vitro-transcribed BC1 RNA (Figure 2B and C, lanes 11 and 12 and lanes 7 and 8, respectively), or in the region located between the two hairpins of the endogenous BC1 RNA (nucleotides 75–127, data not shown). Since the FMRP–BC1 complex controls mRNA translation at synapses (5,6) (Figure 1D and F) we prepared synaptoneurosomes in order to analyse the 2′-O-methylation status of BC1 RNA at synapses. In this case, no 2′-O-methylations were detected on the synaptic BC1 RNA (Figure 2B and C, compare lanes 13, 14 and 9 and 10, respectively, n = 4). The presence of intact BC1 RNA molecules was verified by the presence of a stop of elongation at nucleotide 1 of BC1 RNA (Supplementary Figure S2B). Because BC1 RNA was found differentially 2′-O-methylated in the cell body versus the synapses, we investigated its methylation status in the nucleus. Nuclear and cytoplasmic compartments, used for this analysis, were revealed by the presence and enrichment for dyskerin and Glyceraldehyde 3-phosphate dehydrogenase (Supplementary Figure S1C). As shown in Figure 2D, BC1 RNA from the cytoplasmic or nuclear fractions is 2′-O-methylated (compare lanes 7 and 8 and lanes 9 and 10 for cytoplasmic and nuclear fraction, respectively) underlining that absence of these 2′-O-methylations may have a function at synapses. These findings suggest that BC1 2′-O-methylations occur in nucleus.Figure 2.

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