Surveying Cis-acting sequences of pre-mRNA by adding antisense 2'-o-methyl oligoribonucleotides to a splicing reaction

We chemically synthesized antisense 12 mer 2'-O-methylribonucleotides and surveyed a scanning (signal-tracking) process as well as sequences within a β-globin transcript acting in the splicing reaction in vitro. The pre-mRNA transcript contained the sequences of the first exon, first intron, and a major part of the second exon of the human β-globin gene. We found that the antisense 2'-O-methylribonucleotides could anneal effectively to the target site in the pre-mRNA during the splicing reaction. A 2'-O-methylribonucleotide complementary to the donor (5') splice site completely inhibited authentic splicing and activated an upstream cryptic donor site. A 2'-O-methylribonucleotide complementary to the branch site inhibited normal branch formation and greatly reduced subsequent generation of the spliced product. Six other 2'-O-methylribonucleotides complementary to loci in the exons or the intronic region between the donor and branch sites had no significant effect on the splicing reaction. These observations suggest that an extensive scanning of the present pre-mRNA across the six regions tested is not essential for the splicing reaction. We propose that a short antisense 2'-O-methylribonucleotide provides a practical and convenient method to examine cis-acting sequences of RNA. The advantages of this method in comparison with site-directed mutagenesis or deletion are discussed.