A method for the factor-specific spatio-temporal control of splicing

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in human. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. For instance, Cajal bodies and nuclear speckles function as centers for snRNP assembly and recycling or storage, respectively, whereas the assembly of spliceosomes on nascent pre-mRNAs predominantly occurs in peri-chromatin fibrils. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to any splicing factor by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments. HEK293T single-engineered control cells (ctr) with an N-terminal FKBP-tagged CRISPR knockin PRPF38A or doubly-engineered HEK293T cells with an N-terminal FKBP-tagged CRISPR knockin PRPF38A and stably expressing a lamin A-ECFP-SNAP fusion protein (stbl) were treated with a reversible chemical dimerizer (6 µM rCD1; Treat) or with DMSO as control.