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.
Moore
M J
MJ
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Sharp
P A
PA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D011088:DNA Ligases / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005091:Exons; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013768:Tetrahymena / Q000235:genetics; D014764:Viral Proteins
D012322:RNA Precursors; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D011088:DNA Ligases