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

At synapses, where BC1 is mainly unmethylated, FMRP binds its mRNA targets with higher affinity. (A) Immunoprecipitation of FMRP-associated mRNAs/RNA. Left panel, Western blot of FMRP immunoprecipitated from total brain extracts. Input (1/10), FMRP IP (1/3), the IgG IP (1/3). Right panel, RT–PCR from immunoprecipitated RNA derived from the input (1/10), IgG IP (2/3) and FMRP IP (2/3). The arrow points to the primers used to amplify BC1 RNA (B) Upper panel, low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA (Input, lanes 5 and 6) and immunoprecipitated BC1 RNA (lanes 7 and 8, n = 5). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration. Sequencing reactions are shown in lanes 1–4. Lower panel as upper panel using the BMN297 primer able to detect the 2′-O-methylation on the G56, n = 3. (C) Enrichment of FMRP mRNA targets in immunoprecipitated FMRP complex from both total brain and at synapses. RT–qPCR of FMRP co-immunoprecipitated mRNAs/RNA. Shown is the ratio of the RNA precipitated using FMRP and rabbit unspecific IgGs from total (white histogram) and synaptoneurosomes (grey histogram) brain extracts, each normalized for the respective input. Error bars represent SE: **P < 0.01, Student's test, n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1254-F5: At synapses, where BC1 is mainly unmethylated, FMRP binds its mRNA targets with higher affinity. (A) Immunoprecipitation of FMRP-associated mRNAs/RNA. Left panel, Western blot of FMRP immunoprecipitated from total brain extracts. Input (1/10), FMRP IP (1/3), the IgG IP (1/3). Right panel, RT–PCR from immunoprecipitated RNA derived from the input (1/10), IgG IP (2/3) and FMRP IP (2/3). The arrow points to the primers used to amplify BC1 RNA (B) Upper panel, low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA (Input, lanes 5 and 6) and immunoprecipitated BC1 RNA (lanes 7 and 8, n = 5). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration. Sequencing reactions are shown in lanes 1–4. Lower panel as upper panel using the BMN297 primer able to detect the 2′-O-methylation on the G56, n = 3. (C) Enrichment of FMRP mRNA targets in immunoprecipitated FMRP complex from both total brain and at synapses. RT–qPCR of FMRP co-immunoprecipitated mRNAs/RNA. Shown is the ratio of the RNA precipitated using FMRP and rabbit unspecific IgGs from total (white histogram) and synaptoneurosomes (grey histogram) brain extracts, each normalized for the respective input. Error bars represent SE: **P < 0.01, Student's test, n = 3.

Mentions: To investigate if BC1 2′-O-methylations have an effect on the affinity of FMRP for its mRNA targets, we immunoprecipitated FMRP complexed with BC1 RNA from total brain (Figure 5A, left panel). At first, we monitored that BC1 RNA was indeed associated to FMRP (Figure 5A, right panel), together with a well-known FMRP target (αCaMKII mRNA). The specificity of the interaction is revealed by the absence of D2DR mRNA in the FMRP complex (38). To address whether the BC1 bound to FMRP is 2′-O-methylated or not, we performed low dNTP primer extension assays from FMRP immunoprecipitated RNAs (Figure 5B). Using the BC1-specific primers, BMN155 and BMN297, the 2′-O-methylations were detected in the 5′-stem-loop only in the input RNA (Figure 5B, left and right panels: compare lanes 5 and 6) while the BC1 immunoprecipitated by FMRP was not 2′-O-methylated (Figure 5B: compare lanes 7 and 8). Since the in vitro data showed that FMRP binds less efficiently to 2′-O-methylated BC1 RNA (Figure 3G, Supplementary Figure S3C), we wanted to investigate if the presence or absence of 2′-O-methylations on BC1 RNA had also an effect on stabilizing FMRP target mRNAs in the complex. To address this point we immunoprecipitated FMRP from total brain and synaptoneurosomal extracts and detected the associated mRNAs in the complex. RT–qPCR assays showed a higher co-immunoprecipitation efficiency of FMRP target mRNAs/RNA (αCamKII, Arc, Map1B, BC1) in synaptoneurosome preparations versus total brain (Figure 5C, Supplementary Figure S5). Since at synapses BC1 is mainly detected in the non-methylated form, these findings suggest that absence of 2′-O-methylations favours the FMRP–BC1–mRNA association. Importantly, this complex has been previously shown in vivo to inhibit mRNA translation at synapses (5,6) and Figure 1D and F).Figure 5.


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)

At synapses, where BC1 is mainly unmethylated, FMRP binds its mRNA targets with higher affinity. (A) Immunoprecipitation of FMRP-associated mRNAs/RNA. Left panel, Western blot of FMRP immunoprecipitated from total brain extracts. Input (1/10), FMRP IP (1/3), the IgG IP (1/3). Right panel, RT–PCR from immunoprecipitated RNA derived from the input (1/10), IgG IP (2/3) and FMRP IP (2/3). The arrow points to the primers used to amplify BC1 RNA (B) Upper panel, low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA (Input, lanes 5 and 6) and immunoprecipitated BC1 RNA (lanes 7 and 8, n = 5). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration. Sequencing reactions are shown in lanes 1–4. Lower panel as upper panel using the BMN297 primer able to detect the 2′-O-methylation on the G56, n = 3. (C) Enrichment of FMRP mRNA targets in immunoprecipitated FMRP complex from both total brain and at synapses. RT–qPCR of FMRP co-immunoprecipitated mRNAs/RNA. Shown is the ratio of the RNA precipitated using FMRP and rabbit unspecific IgGs from total (white histogram) and synaptoneurosomes (grey histogram) brain extracts, each normalized for the respective input. Error bars represent SE: **P < 0.01, Student's test, n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1254-F5: At synapses, where BC1 is mainly unmethylated, FMRP binds its mRNA targets with higher affinity. (A) Immunoprecipitation of FMRP-associated mRNAs/RNA. Left panel, Western blot of FMRP immunoprecipitated from total brain extracts. Input (1/10), FMRP IP (1/3), the IgG IP (1/3). Right panel, RT–PCR from immunoprecipitated RNA derived from the input (1/10), IgG IP (2/3) and FMRP IP (2/3). The arrow points to the primers used to amplify BC1 RNA (B) Upper panel, low dNTP concentration primer extension analysis using the BMN155 primer on total brain RNA (Input, lanes 5 and 6) and immunoprecipitated BC1 RNA (lanes 7 and 8, n = 5). The arrows denote 2′-O-methylations at positions G46, C47. For each sample, the second lane shows the lower dNTP concentration. Sequencing reactions are shown in lanes 1–4. Lower panel as upper panel using the BMN297 primer able to detect the 2′-O-methylation on the G56, n = 3. (C) Enrichment of FMRP mRNA targets in immunoprecipitated FMRP complex from both total brain and at synapses. RT–qPCR of FMRP co-immunoprecipitated mRNAs/RNA. Shown is the ratio of the RNA precipitated using FMRP and rabbit unspecific IgGs from total (white histogram) and synaptoneurosomes (grey histogram) brain extracts, each normalized for the respective input. Error bars represent SE: **P < 0.01, Student's test, n = 3.
Mentions: To investigate if BC1 2′-O-methylations have an effect on the affinity of FMRP for its mRNA targets, we immunoprecipitated FMRP complexed with BC1 RNA from total brain (Figure 5A, left panel). At first, we monitored that BC1 RNA was indeed associated to FMRP (Figure 5A, right panel), together with a well-known FMRP target (αCaMKII mRNA). The specificity of the interaction is revealed by the absence of D2DR mRNA in the FMRP complex (38). To address whether the BC1 bound to FMRP is 2′-O-methylated or not, we performed low dNTP primer extension assays from FMRP immunoprecipitated RNAs (Figure 5B). Using the BC1-specific primers, BMN155 and BMN297, the 2′-O-methylations were detected in the 5′-stem-loop only in the input RNA (Figure 5B, left and right panels: compare lanes 5 and 6) while the BC1 immunoprecipitated by FMRP was not 2′-O-methylated (Figure 5B: compare lanes 7 and 8). Since the in vitro data showed that FMRP binds less efficiently to 2′-O-methylated BC1 RNA (Figure 3G, Supplementary Figure S3C), we wanted to investigate if the presence or absence of 2′-O-methylations on BC1 RNA had also an effect on stabilizing FMRP target mRNAs in the complex. To address this point we immunoprecipitated FMRP from total brain and synaptoneurosomal extracts and detected the associated mRNAs in the complex. RT–qPCR assays showed a higher co-immunoprecipitation efficiency of FMRP target mRNAs/RNA (αCamKII, Arc, Map1B, BC1) in synaptoneurosome preparations versus total brain (Figure 5C, Supplementary Figure S5). Since at synapses BC1 is mainly detected in the non-methylated form, these findings suggest that absence of 2′-O-methylations favours the FMRP–BC1–mRNA association. Importantly, this complex has been previously shown in vivo to inhibit mRNA translation at synapses (5,6) and Figure 1D and F).Figure 5.

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