Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.
Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.
Dominski
Z
Z
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Kole
R
R
D001483:Base Sequence; D002874:Chromosome Mapping; D005091:Exons; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D013789:Thalassemia / Q000235:genetics / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D005914:Globins