Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of R70, R111, N104, Y103 to the binding of BC1 RNA. (A–C) Docking models of the FMRP-NT WT, FMRP-NT R70E-R111E and FMRP-NT Y103A-N104A interaction with BC1, respectively. The yellow RNA backbone represents the nucleotides involved in the interaction. The key amino acids detected by mass spectrometry analysis are depicted as blue sticks. (D) EMSA experiment using BC1 RNA, FMRP-NT WT and FMRP-NT R70E-R111E mutant (10–100 ng). Shifted and unbound RNA are indicated by asterisk and arrowhead, respectively; the histogram shows the ratio of the Kd of mutant FMRP-NT versus the Kd of WT FMRP-NT. Error bars represent SE: **P < 0.01, Student's test, n = 3. (E) Competition experiments using unlabelled BC1 (lanes 3–5) and U1 (lanes 6–8) transcripts. (F) and (G) The same as in (D) and (E) but using the Y103A-N104A mutant. P = 0.07 Student's test, n = 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351191&req=5

gkr1254-F4: Effect of R70, R111, N104, Y103 to the binding of BC1 RNA. (A–C) Docking models of the FMRP-NT WT, FMRP-NT R70E-R111E and FMRP-NT Y103A-N104A interaction with BC1, respectively. The yellow RNA backbone represents the nucleotides involved in the interaction. The key amino acids detected by mass spectrometry analysis are depicted as blue sticks. (D) EMSA experiment using BC1 RNA, FMRP-NT WT and FMRP-NT R70E-R111E mutant (10–100 ng). Shifted and unbound RNA are indicated by asterisk and arrowhead, respectively; the histogram shows the ratio of the Kd of mutant FMRP-NT versus the Kd of WT FMRP-NT. Error bars represent SE: **P < 0.01, Student's test, n = 3. (E) Competition experiments using unlabelled BC1 (lanes 3–5) and U1 (lanes 6–8) transcripts. (F) and (G) The same as in (D) and (E) but using the Y103A-N104A mutant. P = 0.07 Student's test, n = 5.

Mentions: The introduction of two alanines at positions 103 and 104 actually removes the hydrogen bonds formed by the wild type residues (compare Figure 4A and C and see Supplementary Table S1). Due to the reduced steric hindrance of the alanine residues, however, the FMRP protein wraps more tightly around BC1 RNA improving other energy terms in the HADDOCK score (i.e. the van der Waals, the buried surface area, the binding and the desolvation energies). The total score therefore decreases from −38.0 in the WT to −49.0 in the mutant, predicting a higher binding affinity. The docking model was experimentally validated by EMSA experiments with the in vitro-transcribed unmodified BC1 RNA and the mutant protein (produced in Escherichia coli; Supplementary Figure S4A). As expected, the apparent Kd decreases by a factor of 2.7 (Figure 4F, strong tendency: P = 0.07, n = 5; apparent Kd FMRP NT WT = 128 ± 22.7 nM and Kd NT Mut = 51 ± 4.1 nM) showing the validity of the modelling. On the other hand the reversion of the positive charges (R70E-R111E, Figure 4A and B) repel the negatively charged phosphates of the RNA backbone, leading to a change in the FMRP orientation towards BC1 and thus to a considerable decrease in the area of interaction surface (Figure 4B). Therefore, the HADDOCK score increases from −38.0 to −25.0, showing a significantly lower affinity. Again the docking prediction was verified by EMSA using the mutant protein (Supplementary Figure S4B). The Kd increased significantly by a factor of 3.8 (Figure 4D, P < 0.01, n = 3. Apparent Kd FMRP NT WT = 128 ± 22.7 nM and Kd FMRP NT Mut = 488 ± 12 nM, respectively). Competition experiments, using both unlabelled BC1 and U1 RNAs, showed that the mutated FMRP-NT/BC1 interaction remains specific (Figure 4E and G). In conclusion the electrostatic interactions of the two arginines R70 and R111 guide the interaction of the second tudor domain with BC1 RNA. Interestingly the binding partners of the two arginines are the phosphates at the 3′-side of the 2′-O-methylated nucleotides G46 and C47.Figure 4.


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)

Effect of R70, R111, N104, Y103 to the binding of BC1 RNA. (A–C) Docking models of the FMRP-NT WT, FMRP-NT R70E-R111E and FMRP-NT Y103A-N104A interaction with BC1, respectively. The yellow RNA backbone represents the nucleotides involved in the interaction. The key amino acids detected by mass spectrometry analysis are depicted as blue sticks. (D) EMSA experiment using BC1 RNA, FMRP-NT WT and FMRP-NT R70E-R111E mutant (10–100 ng). Shifted and unbound RNA are indicated by asterisk and arrowhead, respectively; the histogram shows the ratio of the Kd of mutant FMRP-NT versus the Kd of WT FMRP-NT. Error bars represent SE: **P < 0.01, Student's test, n = 3. (E) Competition experiments using unlabelled BC1 (lanes 3–5) and U1 (lanes 6–8) transcripts. (F) and (G) The same as in (D) and (E) but using the Y103A-N104A mutant. P = 0.07 Student's test, n = 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1254-F4: Effect of R70, R111, N104, Y103 to the binding of BC1 RNA. (A–C) Docking models of the FMRP-NT WT, FMRP-NT R70E-R111E and FMRP-NT Y103A-N104A interaction with BC1, respectively. The yellow RNA backbone represents the nucleotides involved in the interaction. The key amino acids detected by mass spectrometry analysis are depicted as blue sticks. (D) EMSA experiment using BC1 RNA, FMRP-NT WT and FMRP-NT R70E-R111E mutant (10–100 ng). Shifted and unbound RNA are indicated by asterisk and arrowhead, respectively; the histogram shows the ratio of the Kd of mutant FMRP-NT versus the Kd of WT FMRP-NT. Error bars represent SE: **P < 0.01, Student's test, n = 3. (E) Competition experiments using unlabelled BC1 (lanes 3–5) and U1 (lanes 6–8) transcripts. (F) and (G) The same as in (D) and (E) but using the Y103A-N104A mutant. P = 0.07 Student's test, n = 5.
Mentions: The introduction of two alanines at positions 103 and 104 actually removes the hydrogen bonds formed by the wild type residues (compare Figure 4A and C and see Supplementary Table S1). Due to the reduced steric hindrance of the alanine residues, however, the FMRP protein wraps more tightly around BC1 RNA improving other energy terms in the HADDOCK score (i.e. the van der Waals, the buried surface area, the binding and the desolvation energies). The total score therefore decreases from −38.0 in the WT to −49.0 in the mutant, predicting a higher binding affinity. The docking model was experimentally validated by EMSA experiments with the in vitro-transcribed unmodified BC1 RNA and the mutant protein (produced in Escherichia coli; Supplementary Figure S4A). As expected, the apparent Kd decreases by a factor of 2.7 (Figure 4F, strong tendency: P = 0.07, n = 5; apparent Kd FMRP NT WT = 128 ± 22.7 nM and Kd NT Mut = 51 ± 4.1 nM) showing the validity of the modelling. On the other hand the reversion of the positive charges (R70E-R111E, Figure 4A and B) repel the negatively charged phosphates of the RNA backbone, leading to a change in the FMRP orientation towards BC1 and thus to a considerable decrease in the area of interaction surface (Figure 4B). Therefore, the HADDOCK score increases from −38.0 to −25.0, showing a significantly lower affinity. Again the docking prediction was verified by EMSA using the mutant protein (Supplementary Figure S4B). The Kd increased significantly by a factor of 3.8 (Figure 4D, P < 0.01, n = 3. Apparent Kd FMRP NT WT = 128 ± 22.7 nM and Kd FMRP NT Mut = 488 ± 12 nM, respectively). Competition experiments, using both unlabelled BC1 and U1 RNAs, showed that the mutated FMRP-NT/BC1 interaction remains specific (Figure 4E and G). In conclusion the electrostatic interactions of the two arginines R70 and R111 guide the interaction of the second tudor domain with BC1 RNA. Interestingly the binding partners of the two arginines are the phosphates at the 3′-side of the 2′-O-methylated nucleotides G46 and C47.Figure 4.

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