RECOGNITION OF 5' AND 3' SPLICE SITE SEQUENCES IN PRE-MESSENGER-RNA STUDIED WITH A FILTER BINDING TECHNIQUE

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  • 25 August 1987
    • journal article
    • research article
    • Vol. 262  (24) , 11667-11674
Abstract
A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP)fractions, which showed binding activities for both 5'' and 3'' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5'' splice site sequences, but not to a consensus 3'' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5'' splice site, but not a 3'' splice site. The binding activity for the 5'' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5'' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60.degree. C. The binding activity to a 3'' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.