A role for branchpoints in splicing in vivo

Abstract

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from ‘consensus’ sequences for donor and acceptor splice sites by only a few base changes^1–3. Studies in vivo have underlined the importance of these junction nucleotides for splicing^4–7. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing^8–10. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5′ end of the intron is joined via a 2′-5′-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3′ end of the intron, suggests that internal intron sequences may nonetheless be important for splicing^11–13. Indeed, in yeast nuclear genes, the internal sequence 5′-TACTAAC-3′ (or close homologue) is essential for splicing in vivo^14,15. A proposed consensus sequence for branchpoints in mammalian introns is 5′-CT(A/G)A(C/T)-3′ (refs 11, 16, 17). This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5′-CTGACTCTCTCTG-3′)¹¹ greatly enhances the efficiency of splicing of a ‘synthetic’ intron in HeLa cells. A mutated branchpoint sequence, 5′-CTCCTCTCTCTG-3′, in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.