Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O -methyl group was substituted for the 2′-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2′-hydroxyl is not absolutely required at either splice site, the 2′-hydroxyl at the 3′ splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2′-hydroxyl groups in the self-splicing Tetrahymena group I intron.