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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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Related in: MedlinePlus

Functional mapping of the FMRP-NT/BC1 interaction. (A) EMSA using the entire BC1 RNA without (lane 1) or with increasing amounts of FMRP-NT (from 20 to 500 ng, lanes 2–7). The asterisk denotes the shifted FMRP–BC1 complex. Arrowhead denotes unbound BC1 (free). (B) Competition experiment using unlabelled U1 RNA. Lane 1 radiolabelled BC1 RNA, lane 2 radiolabelled BC1 and FMRP-NT (100 ng, asterisk). Lanes 4 and 6 show binding of FMRP-NT and BC1 RNA in the presence of 10–100-fold excess of unlabelled U1 RNA. Lanes 1 and 3 do not contain FMRP-NT. (C) Identification of FMRP residues that are in contact with BC1 RNA. The two tudor domains are underlined in black. The tryptic peptides containing the cross-linked amino acids (highly reactive: E61 and C99, black dots) are shown in blue. (D) Docking model of the interaction of FMRP-NT with the 5′-end of BC1. The backbone of the tudor domains is shown in red, with the two cross-linked peptides highlighted in blue. (E and F) Electrostatic and hydrogen-bond interactions of the FMRP-NT with BC1 RNA. FMRP is shown as a blue ribbon, and the lateral chains of the amino acids involved in the binding network are indicated as blue sticks. The RNA is shown as grey molecular surface with the nucleotides involved in the hydrogen bond and electrostatic network (orange spot), the 2′-O-methylated atoms are shown as a yellow surface. (G) EMSA using FMRP-NT (from 25 to 100 ng) and unmodified BC1 RNA (lanes 2–5) or 2′-O-methylated BC1 RNA (lanes 7–10). Lanes 1 and 6, unbound BC1 RNA. The asterisk indicates the FMRP-NT/BC1 RNA complex. The histogram shows the ratio of the Kd of 2′-O-methylated BC1 construct versus the Kd of unmodified BC1 construct. Error bars represent SE: **P < 0.01, Student's test, n = 3.
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gkr1254-F3: Functional mapping of the FMRP-NT/BC1 interaction. (A) EMSA using the entire BC1 RNA without (lane 1) or with increasing amounts of FMRP-NT (from 20 to 500 ng, lanes 2–7). The asterisk denotes the shifted FMRP–BC1 complex. Arrowhead denotes unbound BC1 (free). (B) Competition experiment using unlabelled U1 RNA. Lane 1 radiolabelled BC1 RNA, lane 2 radiolabelled BC1 and FMRP-NT (100 ng, asterisk). Lanes 4 and 6 show binding of FMRP-NT and BC1 RNA in the presence of 10–100-fold excess of unlabelled U1 RNA. Lanes 1 and 3 do not contain FMRP-NT. (C) Identification of FMRP residues that are in contact with BC1 RNA. The two tudor domains are underlined in black. The tryptic peptides containing the cross-linked amino acids (highly reactive: E61 and C99, black dots) are shown in blue. (D) Docking model of the interaction of FMRP-NT with the 5′-end of BC1. The backbone of the tudor domains is shown in red, with the two cross-linked peptides highlighted in blue. (E and F) Electrostatic and hydrogen-bond interactions of the FMRP-NT with BC1 RNA. FMRP is shown as a blue ribbon, and the lateral chains of the amino acids involved in the binding network are indicated as blue sticks. The RNA is shown as grey molecular surface with the nucleotides involved in the hydrogen bond and electrostatic network (orange spot), the 2′-O-methylated atoms are shown as a yellow surface. (G) EMSA using FMRP-NT (from 25 to 100 ng) and unmodified BC1 RNA (lanes 2–5) or 2′-O-methylated BC1 RNA (lanes 7–10). Lanes 1 and 6, unbound BC1 RNA. The asterisk indicates the FMRP-NT/BC1 RNA complex. The histogram shows the ratio of the Kd of 2′-O-methylated BC1 construct versus the Kd of unmodified BC1 construct. Error bars represent SE: **P < 0.01, Student's test, n = 3.

Mentions: The N-terminus of FMRP (FMRP-NT; residues 1–217) contains two tudor domains (10), has RNA-binding properties (8,48), and interacts directly with the BC1 5′-hairpin (5,9). Because FMRP-NT tends to aggregate (48) (and data not shown) we tested its correct folding via the binding to the entire unmodified BC1 RNA (Figure 3A and B) as well as to poly riboG and poly riboC (data not shown). The FMRP-NT binds BC1 RNA with high affinity (apparent Kd = 128 ± 22.7 nM, the same within experimental error as in ref. (9); see Figure 3A for the gel and Supplementary Figure S3A for the Kd plot). Furthermore, a similarly structured RNA, U1 snRNA, was unable to compete with BC1 RNA for the FMRP binding (Figure 3B, compare lane 2 with lanes 4, 5 and 6).Figure 3.


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)

Functional mapping of the FMRP-NT/BC1 interaction. (A) EMSA using the entire BC1 RNA without (lane 1) or with increasing amounts of FMRP-NT (from 20 to 500 ng, lanes 2–7). The asterisk denotes the shifted FMRP–BC1 complex. Arrowhead denotes unbound BC1 (free). (B) Competition experiment using unlabelled U1 RNA. Lane 1 radiolabelled BC1 RNA, lane 2 radiolabelled BC1 and FMRP-NT (100 ng, asterisk). Lanes 4 and 6 show binding of FMRP-NT and BC1 RNA in the presence of 10–100-fold excess of unlabelled U1 RNA. Lanes 1 and 3 do not contain FMRP-NT. (C) Identification of FMRP residues that are in contact with BC1 RNA. The two tudor domains are underlined in black. The tryptic peptides containing the cross-linked amino acids (highly reactive: E61 and C99, black dots) are shown in blue. (D) Docking model of the interaction of FMRP-NT with the 5′-end of BC1. The backbone of the tudor domains is shown in red, with the two cross-linked peptides highlighted in blue. (E and F) Electrostatic and hydrogen-bond interactions of the FMRP-NT with BC1 RNA. FMRP is shown as a blue ribbon, and the lateral chains of the amino acids involved in the binding network are indicated as blue sticks. The RNA is shown as grey molecular surface with the nucleotides involved in the hydrogen bond and electrostatic network (orange spot), the 2′-O-methylated atoms are shown as a yellow surface. (G) EMSA using FMRP-NT (from 25 to 100 ng) and unmodified BC1 RNA (lanes 2–5) or 2′-O-methylated BC1 RNA (lanes 7–10). Lanes 1 and 6, unbound BC1 RNA. The asterisk indicates the FMRP-NT/BC1 RNA complex. The histogram shows the ratio of the Kd of 2′-O-methylated BC1 construct versus the Kd of unmodified BC1 construct. Error bars represent SE: **P < 0.01, Student's test, n = 3.
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Related In: Results  -  Collection

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gkr1254-F3: Functional mapping of the FMRP-NT/BC1 interaction. (A) EMSA using the entire BC1 RNA without (lane 1) or with increasing amounts of FMRP-NT (from 20 to 500 ng, lanes 2–7). The asterisk denotes the shifted FMRP–BC1 complex. Arrowhead denotes unbound BC1 (free). (B) Competition experiment using unlabelled U1 RNA. Lane 1 radiolabelled BC1 RNA, lane 2 radiolabelled BC1 and FMRP-NT (100 ng, asterisk). Lanes 4 and 6 show binding of FMRP-NT and BC1 RNA in the presence of 10–100-fold excess of unlabelled U1 RNA. Lanes 1 and 3 do not contain FMRP-NT. (C) Identification of FMRP residues that are in contact with BC1 RNA. The two tudor domains are underlined in black. The tryptic peptides containing the cross-linked amino acids (highly reactive: E61 and C99, black dots) are shown in blue. (D) Docking model of the interaction of FMRP-NT with the 5′-end of BC1. The backbone of the tudor domains is shown in red, with the two cross-linked peptides highlighted in blue. (E and F) Electrostatic and hydrogen-bond interactions of the FMRP-NT with BC1 RNA. FMRP is shown as a blue ribbon, and the lateral chains of the amino acids involved in the binding network are indicated as blue sticks. The RNA is shown as grey molecular surface with the nucleotides involved in the hydrogen bond and electrostatic network (orange spot), the 2′-O-methylated atoms are shown as a yellow surface. (G) EMSA using FMRP-NT (from 25 to 100 ng) and unmodified BC1 RNA (lanes 2–5) or 2′-O-methylated BC1 RNA (lanes 7–10). Lanes 1 and 6, unbound BC1 RNA. The asterisk indicates the FMRP-NT/BC1 RNA complex. The histogram shows the ratio of the Kd of 2′-O-methylated BC1 construct versus the Kd of unmodified BC1 construct. Error bars represent SE: **P < 0.01, Student's test, n = 3.
Mentions: The N-terminus of FMRP (FMRP-NT; residues 1–217) contains two tudor domains (10), has RNA-binding properties (8,48), and interacts directly with the BC1 5′-hairpin (5,9). Because FMRP-NT tends to aggregate (48) (and data not shown) we tested its correct folding via the binding to the entire unmodified BC1 RNA (Figure 3A and B) as well as to poly riboG and poly riboC (data not shown). The FMRP-NT binds BC1 RNA with high affinity (apparent Kd = 128 ± 22.7 nM, the same within experimental error as in ref. (9); see Figure 3A for the gel and Supplementary Figure S3A for the Kd plot). Furthermore, a similarly structured RNA, U1 snRNA, was unable to compete with BC1 RNA for the FMRP binding (Figure 3B, compare lane 2 with lanes 4, 5 and 6).Figure 3.

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