Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction.
The second intron (betaIVS-II) of the human beta-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 betaIVS-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 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 betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-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 betaIVS-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 betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-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.
Antoniou
M
M
Department of Experimental Pathology, UMDS, Guy's Hospital, London Bridge, London SE1 9RT, UK. m.antoniou@umds.ac.uk
Geraghty
F
F
Hurst
J
J
Grosveld
F
F
D000818:Animals; D001483:Base Sequence; D005091:Exons; D005786:Gene Expression Regulation / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D005914:Globins