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Nucleic Acids Research, 1992, Vol. 20, No. 17 4457-4464
© 1992

MOLECULAR BIOLOGY

Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing

Silivia M.L. Barabino+, Brian S. Sproat and Angus I. Lamond*

*To whom correspondence should be addressed

Received June 15, 1992. Revised August 14, 1992. Accepted August 14, 1992.

Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop IIa region of U2 snRNA (nucleotides 54–72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57–68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.

+Present address: DIBIT H S.Raffaele, via Olgettina 60, 20132 Milan, Italy


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