An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

Abstract U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

value of the isolated Sm B-D 3 . The reference spectra and the spectra recorded after saturation of the amide protons are depicted in B and C, respectively. The intensity ratio between B and C determines the value of the { 15 N-1 H} heteronuclear NOE. When the value is close to 1, the residue is considered as rigid while a value lower than 0.25 supports flexibility. All the residues that show low values of { 15 N-1 H} heteronuclear NOE remain visible in U1 snRNP. Thus, the flexible parts of SmB-D 3 remain visible in U1 snRNP. (blue). In B and C, SmB-D 3 is not labelled and therefore invisible on the 2D 15 N-1 H HSQC spectra. The blue spectrum corresponds to the folded of U1-C and it overlaps very well with the spectra of full length U1-C. The additional resonances observed on the U1-C spectrum (red) correspond to the C-terminal tail of U1-C and the signals are in the centre of the spectrum, in the random coiled region. (D) Overlay of the 2D 15 N-1 H HSQC TROSY of U1-C in complex with SmB-D 3 (black) or in U1 snRNP (red). The observed NMR signals in the context of U1 snRNP correspond to the C-terminal tail while the signals from the zinc finger domain, that take part of the core of the particle, are broadened and bleached from the NMR spectra. ORFs were cloned in fusion with a C-terminal hexa-histidine tag using the pET28a plasmid.
Purification of the U1 snRNP protein components. Sm B/B'-D3 was solubilized in buffer A (Hepes 20 mM pH 7.8, NaCl 1 M, Urea 2 M, -mercapto-ethanol 2.8mM) in presence of 10ug/ml of DNAseI and lysozyme. The cells were lysed using a microfluidizer using 5 consecutive cycles at 15,000 psi. The cell lysate was clarified by centrifugation (30,000g at 4°C during 45 minutes) and loaded at 0.5 ml/min on a 5 ml HisTrap column (GE Healthcare) at 4°C.
The column was washed with buffer A (100 ml) and 10% B (50 ml) and eluted with buffer B (Hepes 20 mM pH 7.8, NaCl 1 M, imidazole 500 mM,  -mercapto-ethanol 2.8 mM). The heterodimer Sm B-D3 was cleaved by thrombin (Sigma) and dialyzed in buffer C (sodium phosphate 10 mM pH6.0, DTT 5mM). The sample was then loaded on the HiTrap SP 5ml column, washed with buffer C and eluted with buffer D (sodium phosphate 10 mM pH6.0, 2 M NaCl, DTT 5mM). Finally, the protein was dialyzed with buffer E (sodium phosphate 10 mM pH6.0, DTT 5mM) and loaded on a size exclusion column (SEC) previously equilibrated with buffer E. The protein was concentrated to 100 µM and stored at -80°C. M, imidazole 300 mM). The protein was cleaved by TEV protease at room temperature and diluted 5 times to reduce the NaCl concentration to 100 mM. The cleavage reaction was loaded on a 5 ml HiTrap SP column (GE Healthcare) at room temperature previously equilibrated with buffer P (Hepes 20 mM pH 7.5, NaCl 0.1 M), washed with buffer P and U1-A was eluted with buffer Q (Hepes 20 mM pH 7.5, NaCl 2 M), concentrated and further purified by SEC in buffer R (sodium-phosphate buffer 10 mM pH 6.8, NaCl 50 mM, EDTA 0.5 mM, DTT 5 mM). The protein was concentrated to 100 µM and stored at -80°C.
U1-C was solubilized in buffer S (Hepes 10 mM pH 7.8, NaCl 0.1 M, Urea 0.5 M, -mercaptoethanol 2.8 mM) in presence of 10ug/ml of DNAseI and lysozyme. The cells were lysed using a microfluidizer using 5 consecutive cycles at 15,000 psi. The cell lysate was clarified by centrifugation (30,000g at 4°C during 45 minutes) and loaded on a 5 ml HisTrap column (GE Healthcare) at 4°C. The column was washed with buffer S, 10% buffer T (Hepes 10 mM pH 7.8, NaCl 0.1 M, Urea 0.5 M, Imidazole 250 mM, -mercapto-ethanol 2.8 mM) and U1-C was eluted with 100% buffer T. The protein was concentrated at 4°C and further purified by SEC in buffer U (sodium-phosphate buffer 10 mM pH 6.8, NaCl 50 mM, DTT 5 mM). Elution fractions containing ~10 M of U1-C were flash frozen in liquid nitrogen and stored at -80°C.
Cloning of the plasmid allowing the transcription of U1 snRNA and its derivative. A plasmid (pUC19-U1 snRNA) containing a T7 promoter followed a Hammerhead ribozyme fused to the U1 snRNA was ordered (Genscript). The plasmid was linearized by SalI. The construct is described in Supplementary Figure 2.