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 changes1–3. Studies in vivo have underlined the importance of these junction nucleotides for splicing4–7. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing8–10. 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 splicing11–13. Indeed, in yeast nuclear genes, the internal sequence 5′-TACTAAC-3′ (or close homologue) is essential for splicing in vivo14,15. 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.