Recruitment of a splicing factor to the nuclear lamina for its inactivation

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 humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio temporal dynamics participate in the regulation of splicing. 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 in HEK293T cells 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 many splicing factors 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 environment

A multi factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification mass spectrometry and RNA UV crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock down of C9ORF78 leads to alternative NAGNAG 3 splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3 splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late stage splicing regulatory protein that takes advantage of a multi factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicin

Branch point strength controls species specific CamK2 amp; 946; alternative splicing and regulates LTP

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