Processing of the mouse beta-globin mRNA precursor: at least two cleavage-ligation reactions are necessary to excise the larger intervening sequence.

The β-globin mRNA precursor contains one copy of mRNA that is divided into three segments by two intervening sequences (IVS) (Smith and Lingrel, 1978; Kinniburgh, Mertz and Ross, 1978; Tilghman et al., 1978a). We have investigated the intracellular processing pathway of the 1800 nucleotide precursor by analyzing the organization of mRNA segments and intervening sequences in two classes of processing intermediates, one containing 1030 and the other 900 nucleotides. These RNAs were purified from pulse-labeled erythroid cells so that each class could be analyzed separately, thereby allowing us to derive a probable processing scheme and to compare the rates of each cleavage step. The 1030 nucleotide intermediate is 700–800 nucleotides shorter than the precursor and contains two intervening sequences. This RNA is thus generated by excision from the precursor of a major portion of the larger IVS. The 900 nucleotide RNA contains two structurally distinct molecules. One of the 900 nucleotide RNAs still contains the two IVS. The other 900 nucleotide RNA contains only one IVS derived from what was initially the larger IVS. The smaller IVS has been completely excised from this RNA to yield a spliced RNA segment derived from the 5′ terminal and middle mRNA fragments. The fully spliced 790 nucleotide β-globin mRNA is generated by excision of the remaining IVS from the 900 nucleotide RNAs. These data are consistent with a stepwise elimination of the larger IVS by at least two cleavage-ligation reactions. This result implies that the new nucleotide sequence arrangement generated by the first cleavage-ligation reaction is crucial to the precise joining of the mRNA coding regions during the final processing step.