Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction

Abstract

The second intron (βIVS-II) of the human β-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3′-end formation. This report presents quantitative comparisons between βIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a β-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3′-end formation and in the absence of exon skipping. Therefore, 3′ cleavage of β-globin transcripts requires the presence of βIVS-II sequences, but not the splicing reaction. The placement of βIVS-II in the IVS-I position did not reduce the efficiency of 3′ cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of βIVS-I in the intron II position, reduced the efficiency of 3′-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns βIVS-I or β-globin IVS-II, only partially substitute (16 and 30% respectively) for βIVS-II. Hybrid introns show that efficient 3′-end formation is strongly enhanced by the presence of the terminal 60 nt of βIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3′-end formation and may act as a post-transcriptional regulatory step in gene expression.