pre-mRNA splicing · Coordinated regulation of transcription and pre-mRNA splicing at gene promoters

Accurate pre-mRNA splicing is a fundamental step for gene expression. SR proteins, a protein family of splicing regulators, play an essential role in the process by defining intron-exon boundaries and recruiting the spliceosome to carry out splicing. Pre-mRNA splicing occurs co-transcriptionally and it is now emerging that promoter identity modulates alternative splicing, possibly through the recruitment of SR proteins or through kinetic coupling by altering Pol II elongation rates. The aim of the present proposal is to investigate the relationship between promoter elements, differential SR protein recruitment to genes and splicing outcome.The genome-wide recruitment of SR proteins to promoter elements will be investigated resorting to innovative approaches. SR proteins tagged with LAP-tag and expressed in ""BAC cell lines"" will be isolated with associated DNA sequences (ChIP) which will then be used to interrogate a genome-wide promoter microarray (ChIP-on-CHIP). This will reveal similarities and differences in recruitment to genes and gene regions among SR protein family. To test how promoter elements influence spliceosome assembly and splicing outcome, model-genes containing alternative and constitutively spliced exons will be created. The focus will be on genes relevant for neuronal differentiation. Model genes will be introduced in cells already expressing individual SR proteins BACs, creating ""Double-BAC cell lines"". The results expected from this project will provide a global view of SR protein role in regulating and coupling transcription and pre-mRNA splicing. Elucidating these regulatory mechanisms is key for the understanding of genomic and proteomic diversity.""