EFFICIENT 3'-END FORMATION OF HUMAN BETA-GLOBIN MESSENGER-RNA IN-VIVOREQUIRES SEQUENCES WITHIN THE LAST INTRON BUT OCCURS INDEPENDENTLY OFTHE SPLICING REACTION

The second intron (beta IVS-II) of the human beta-globin gene is essen tial for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantit ative comparisons between beta IVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which fu rther defines it's function. In marked contrast to a beta-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 beta-globin transcripts requires the presence of beta IVS -II sequences, but not the splicing reaction. The placement of beta IV S-II in the IVS-I position did not reduce the efficiency of 3' cleavag e indicating that the distance between the necessary element(s) in thi s intron and the polyadenylation recognition site is not a crucial fac tor. Subsequent placement of beta IVS-I in the intron II position, red uced the efficiency of 3'-end formation to only 16% of normal. A direc t replacement of intron II by the heterologous introns beta IVS-I or a lpha-globin IVS-II, only partially substitute (16 and 30% respectively ) for beta IVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of beta IV S-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency o f 3'-end formation and may act as a post-transcriptional regulatory st ep in gene expression.