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Nucleic Acids Research, 1990, Vol. 18, No. 24 7373-7379
© 1990

Articles

Sequence-specific affinity selection of mammalian splicing complexes

Ursula Ryder, Brian S. Sproat and Angus I. Lamond*

European Molecular Biology Laboratory Meyerhofstrasse 1, Postfach 102209, D6900 Heidelberg 1, FRG

*To whom correspondence should be addressed

Received August 17, 1990. Revised November 1, 1990. Accepted November 1, 1990.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/blotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.


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