Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA

Abstract

An RNase A protection assay was employed to investigate the interaction of nuclear components with a precursor-mRNA derived from the adenovirus 2 major late transcription unit in a splicing extract from HeLa cells. Upon incubation in the extract, two regions in the precursor-RNA become resistant to digestion with RNase A. After short incubation times (5 min) at 30 °C, fragments mapping upstream from the branch point in the intron are obtained. After ten minutes or more, additional oligonucleotides, derived from the 5′ splice site, are protected. RNase A protection of different RNA substrates demonstrates that a 5′ splice site is not required for the binding of components to the branch point region. For interaction with this site, the polypyrimidine stretch just upstream from the 3′ splice site is essential. Binding to the 5′ splice site occurs only in the presence of an intact 3′ end of the intron. Preincubation of the extract with excess unlabelled RNA containing only a 3′ splice site leads to efficient competition of binding, both in the branch point region and at the 5′ splice site, whereas an RNA that contains only 5′-splice-site sequences has no effect on the interaction with the mRNA precursor. This indicates that stable association with the 5′ splice site requires prior binding of components in the branch point region. When splicing complexes are digested with RNase A, it becomes apparent that only the branch point region is sequestered into a ribonucleoprotein (RNP) structure in the 35 S complex. The 5′ splice site becomes resistant to RNase A only when a 50 S splicing complex has been assembled. Degradation of specific regions in U1, U2 and U4 RNA with complementary oligodeoxynucleotides and RNase H has been used to analyse involvement of the U small nuclear RNPs (snRNPs) in the protection reaction. The 5′ end of U2 RNA is essential for protection of the branch point region. RNA sequences in a loop of U2 RNA (nucleotides 65 to 78) are required for the formation of an RNase-A-resistant structure at the 5′ splice site. Taken together, these results suggest that U2 snRNP participates in the formation of a pre-splicing complex, the 5′ end of its RNA being involved in the observed binding. Conversion to a 50 S splicing complex is obtained after the binding of Ul and U4/U6 snRNPs, which also requires sequences in a loop of U2 RNA. Possible interactions between the individual snRNPs and between snRNPs and precursor-mRNA are discussed.