1. Howard Hughes Medical Institute,
2. Laboratory of Molecular Neuro-Oncology,
3. Biocomputing, Information Technology, and,
4. Laboratory of Computational Genomics, The Rockefeller University, 1230 York Ave,, New York, New York 10021, USA
5. Banting and Best Department of Medical Research, University of Toronto, Ontario M5G 1L6, Canada
6. These authors contributed equally to this work.
7. Present addresses: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK (J.U.); Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511, USA (G.S.); UCSD, Scripps Institution of Oceanography, Scripps Genome Center, 8750 Biological Grade, La Jolla, California 92037, USA (B.T., T.G.); Eastern Mediterranean University, Famagusta, Cyprus (B.T.).

Correspondence to: Robert B. Darnell^1,2 Correspondence and requests for materials should be addressed to R.B.D. (Email: darnelr@rockefeller.edu).

Abstract

Nova proteins are neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova's effect to inhibit or enhance exon inclusion, which led us to examine the relationship between the map and Nova's mechanism of action. Nova binding to an exonic YCAY cluster changed the protein complexes assembled on pre-mRNA, blocking U1 snRNP (small nuclear ribonucleoprotein) binding and exon inclusion, whereas Nova binding to an intronic YCAY cluster enhanced spliceosome assembly and exon inclusion. Assays of splicing intermediates of Nova-regulated transcripts in mouse brain revealed that Nova preferentially regulates removal of introns harbouring (or closest to) YCAY clusters. These results define a genome-wide map relating the position of a cis-acting element to its regulation by an RNA binding protein, namely that Nova binding to YCAY clusters results in a local and asymmetric action to regulate spliceosome assembly and alternative splicing in neurons.

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