Independent Removal of Two Identical or Different Introns from mRNA Precursors In Vitro1

In vitro splicing of 11 different RNA transcripts carrying two introns was performed to determine whether the removal of an intron was dependent on its position or removal of the other intron. The original RNA transcript carried two identical units, each consisting of the first exon-first intron-a major part of the second exon of the human beta-globin gene. Five transcripts carrying a mutation in one of the two introns of the original RNA and one carrying mutations in both introns were prepared. One of the other RNA transcripts had a shorter middle exon than did the original transcript. The remaining three RNAs had at least one shortened form of the human beta-globin second intron. The results can be summarized as follows. 1) There was no obligatory order in the excision of the two identical or different introns, although the 5' proximal intron may be removed more rapidly in some cases. The efficiency of removal of the 5' proximal intron seems to be higher than or similar to that of the distal intron. 2) Excision of a mutated intron was inhibited, but no mutation significantly affected the excision of the other intact intron. 3) Except in an RNA transcript with two mutated introns, significant splicing between exon 1 and exon 3 (exon jumping) did not take place. 4) These results support the proposal that the two introns are basically independent of each other as to their removal.