A method to study alternative splicing using solution NMR spectroscopy
A recent "Nucleic Acid Research" paper by the Allain group (IBC) in collaboration with the Cryo-EM hub (CEMK) and PSI shows that it is possible to reconstitute U1 snRNP in vitro, study its dynamic parts in solution and interactions with protein partners controlling alternative splicing.
Altered RNA metabolism has been linked with numerous diseases. Therefore, understanding and correcting RNA processing is a major future challenge towards the development of artificial RNA processing effectors. U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA splicing 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 method to reconstitute U1 snRNP in vitro using mostly full-length components in order to study some of the invisible parts of the spliceosome using NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle (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 interaction between the splicing factor PTBP1 and U1 snRNP and mapped the interaction surfaces. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling RNA splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.
Link to the paper in external pageNucleic Acid Research.