5' splice site mutations in human diseases: mechanisms and antisense therapy

Cold Spring Harbor Laboratory, Cold Spg Hbr NY

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Abstract

DESCRIPTION (provided by applicant): At least 15% of all disease-associated point mutations affect pre-mRNA splicing. Mutations at the 5' splice site (5'ss) are associated with a variety of human diseases, and are especially detrimental, due to their interference with initial recognition of the exon-intron boundary by U1 snRNP (a spliceosomal component). While some 5'ss can tolerate point mutations, others cannot, and these result in aberrant splicing that may cause human diseases; yet, the reasons for this difference are poorly understood. The objective of the proposed research is to enhance our understanding of 5'ss recognition and determine the characteristics of 5'ss that are prone to perturbation by mutations. The mechanism of splicing modulation by antisense oligonucleotides will also be examined, so as to facilitate their development as targeted therapeutics. Aim one will identify general rules that determine 5'ss susceptibility to mutations, focusing on one of the 5'ss in BRCA2 that is mutated in human breast cancers. Mutational analysis within this 5'ss will be performed to map positions critical fo its proper recognition. Other disease-related genes with exactly the same 5'ss motif will be studied to determine the generality of the findings. By establishing the types of 5'ss susceptible to mutations, we can predict individuals at risk of developing certain diseases, and improve targeted therapeutics. Less than 1% of introns are of the GC-AG type, which has long been assumed to be spliced in the same manner as the canonical GT-AG type. Yet, T?C mutations at the +2 position in GT-AG introns abolish recognition, suggesting a different mechanism or snRNP may be necessary for normal GC-AG recognition. Using biochemical and molecular techniques to manipulate the alternative usage of GC and GT 5'ss in NPHP1 (nephoronophthisis 1), aim two will either validate the current assumptions or uncover a new mechanism for GC 5'ss recognition. Finally, antisense oligonucleotides (ASOs) are a promising therapeutic approach to correct splicing in defective genes. Yet, developing effective ASOs is slow and labor-intensive, and the underlying mechanisms are not fully understood. Aim three will use biochemical and bioinformatics approaches to dissect a potential mechanism of action of ASOs that alter splicing and determine whether the ASOs can structurally stabilize U1 snRNP binding to 5'ss. Altogether, these studies will explore basic mechanisms to contribute to the understanding of disease-causing 5'ss mutations, to improve predictions of disease-causing splicing mutations, and to facilitate targeted-ASO drug development. The technical and conceptual experience gained by completing this proposed research will be critical steps towards achieving my long-term career goal to become an independent biomedical researcher.