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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

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.


Primer Labelling
Each primer was [32P] γATP labelled according to the standard procedures. Radiolabelled primers were purified on BioRad Spin 6 columns according to the manufacturer's instructions.

In vitro transcription
Sense BC1 and U2 RNAs were generated using the T7-directed in vitro transcription (MegaScriptT7 kit, Ambion) and pBCX607 (gift of H.Tiedge) and pT7U2 (gifts of T.Kiss) plasmids respectively.
For the sense U1, the human U1 gene was amplified using the plasmid phU1 (gift of T. Kiss) and the U1 5'T7 and U1 3' primers. The PCR product was used as template to transcribe full length U1 sense (MegaScriptT7 kit, Ambion). For the 3' hairpin BC1 transcripts, the BC1 3'end was amplified using the pBCX607. Transcripts were purified on 8 M urea-8% acrylamide gels.

RNA isolation
RNA from total brain tissue was extracted with Trizol (Invitrogen) and separated on an 8 M urea/8% acrylamide gel. The band corresponding to BC1 RNA was visualized by UV shadowing, excised, eluted overnight in 200 µl elution buffer (500mM NaOAc pH 5.3, 1 mM EDTA), extracted with phenol-chloroform isoamyl alcohol, and precipitated using absolute ethanol and glycogen. RNA was pelleted and washed using 70% EtOH and dried. RNA enriched for BC1 was purified by gel electrophoresis using in vitro synthesized BC1 RNA as a marker. After purification, the identity of the RNA was confirmed by Northern blotting using the radiolabelled CD22 DNA primer.

Reconstruction of BC1 RNA
The modified or unmodified 5' hairpin was aligned to the 3' hairpin using an oligo DNA splint and ligated in the presence of T4 RNA ligase as previously described (1). The final product was visualized on an 8 M-urea/10% acrylamide gel and the corresponding band cut, eluted and radiolabelled as described before.

RT-qPCR (SYBR-Green and TaqMan)
For total brain cytoplasmic extracts (WT and BC1 KO) equal volume from each polysomal fraction was extracted and the RNA was reverse transcribed by MoMLV reverse transcriptase (Invitrogen). RT-qPCR analysis of polysomal gradients from WT and BC1 KO brain was performed using the TaqMan (Applied Biosystems) system while from synaptosomal extracts using a LightCycler480 (Roche).

Western blot analysis
Protein analysis was performed as previously described (2). In this case 5 µg of total and synaptic proteins were analyzed. Synaptic preparations were evaluated by the presence of synaptic APP (3) and enrichment of the postsynaptic protein PSD-95 (4). For quantitative western blot, chemilumescence signal was acquired using the Fujifilm LAS3000 and further quantified using the Image Quant software (GE Healthcare) or AIDA software (Raytest Isotopenmeßgeräte GmbH). Western blotting was performed following standard procedures, using precast 18-well 4-12% Bis-Tris CriterionXT gels from BioRad, run in 1X MOPS.

Synaptoneurosome preparation and RNA extraction
Synaptoneurosomes were prepared by homogenizing fresh cortex-hippocampus-striatum tissue in ice-cold buffer and processed through a flotation Percoll gradient as previously described (2). The synaptoneurosomes were resuspended in 0.5 ml HEPES-Krebs buffer (20 mM HEPES pH 7.4, 147 mM NaCl, 3 mM KCl, 10 mM glucose, 2 mM MgSO4, 2 mM CaCl2), equilibrated at 37°C for 10 min, and then lysed for RNA and protein analysis.
Synaptosomal RNA was extracted using proteinase K. Briefly, synaptosomes were lysed in 0.2 M NaOAc pH 4.8, 0.5% SDS, and 1 mM EDTA; 0.2 mg/ml proteinase K was added and digestion continued for 30 min at 37°C. A phenol/CHCl 3 extraction was performed, followed by CHCl 3 extraction and precipitation in 2.5 vol EtOH at -80°C overnight with 10 µg of glycogen added as a carrier. RNA was quantified and evaluated with an ND-1000 spectrophotometer (NanoDrop Technologies) as well as by electrophoresis.

RT-PCR and RT-qPCR analysis
First-strand synthesis was performed using p(dN)6 and 100 U of M-MLV RTase (Invitrogen).
PCR was performed as previously described (7) using specific oligonucleotides to amplify β-Actin, αCaMKII, Arc and Map1B using oligos described in Supplementary Information. Real Time PCR was performed using an ABI 7300 Sequence Detector with dual-labeled TaqMan probes (Applied Biosystems) using Pre-Developed TaqMan assays, as described above.

Sucrose gradient to separate polysomes and mRNPs
Each gradient was collected in 10 fractions followed by the addition of 1% SDS (final concentration), 80 pg of synthetic rpL22 or 25 pg of synthetic BC200 RNAs, 10 µg glycogen, and proteinase K (100 µg/ml) and incubated for 30 min at 37°C. The synthetic RNAs were used to normalize for possible RNA loss during RNA phenol/chloroform extraction and precipitation from each fraction. RNAs were precipitated with 0.2 M NaOAc pH 4.8 and 0.7 6 vol of isopropanol. The pellets were resuspended in 30 µl or 15 µl of ddH 2 O for total brain or synaptosome respectively, and the RNA fractions 1-5 (polysomal fraction, P) and 6-10 (mRNP fraction, NP) were pooled and RNA quality/quantity was assessed by 1.8% agarose formaldehyde gel electrophoresis and spectrophotometry (ND-1000 spectrophotometer, Nanodrop Technology). mRNAs of interest were quantified by RT-qPCR (see below), and the percentage of mRNAs on polysomes (PMP) was calculated as the ratio of the mRNA in fraction P over the total of fractions P and NP; a higher value indicates that a higher portion of the mRNA was associated with ribosomes at the time of fractionation and thus engaged in translation.

Computational procedures
The program generates different fragments of RNA that are further iteratively assembled applying constraints on the basis of the 2D structure. One hundred 3D structures of BC1 5' end were generated and for each of them a steepest descent energy minimization has been performed in order to refine the model. The structure with the lowest free energy value was For the interaction analysis, the complex with the best HADDOCK score was selected from the most populated families of each docking simulations.
Currently no docking program is able to consider RNA 2'-O-methylations in the interaction prediction, so the best HADDOCK score has been evaluated for the WT (unmethylated) BC1 7 5' complexes and then two methyl groups on both G46 and C47 (Gm46/Cm47), have been added by molecular modelling. The methyl groups on the 2'-O atom of the ribose moieties, and the structural differences caused by the amino acid substitution were modelled using the Sybyl 6.0 program (http://www.tripos.com) and the resulting structures were manually checked for altered interactions. The analysis of hydrogen bonds and electrostatic interactions were performed with the WHAT IF (9) and VMD (10) programs, respectively.