Investigation of the mechanism of pre-mRNA splicing using phosphorothioate analogues of RNA
The Sp diastereoisomer of adenosine 5'-O-(l-thiotriphosphate) (ATPaS) was found to be incorporated into RNA transcribed by T7 RNA polymerase with an apparent KM similar to that for ATP; the Rp diastereoisomer was neither a substrate nor a competitive inhibitor. The configuration of the thiophosphodiester link in the RNA produced was analysed with stereospecific nucleases. Surprisingly, the nucleases exhibited reduced discrimination compared to their activity on dinucleotides. The results show that phosphorothioate linkages in T7 RNA polymerase transcripts are in the Rp configuration. Thus, the transcription reaction proceeds with inversion of configuration at phosphorus. In vitro transcription by T7 RNA polymerase in the presence of a nucleoside 5'-O-(1-thiotriphosphate) has been used to prepare pre-mRNA analogues of the small intron of a rabbit ?-globin gene and flanking exon sequences. Incubation of transcripts prepared with ATPaS in a HeLa cell nuclear extract showed that the presence of the thionucleotide in a transcript inhibited splicing, but a novel product (termed E2*) was formed by cleavage three nucleotides upstream of the 3' splice site. This product was formed with the same kinetics as the intermediates of a normal splicing reaction, and its formation depended on the presence of ATP, Mg2+, and intact small nuclear RNAs U1, U2, and U6. Hence, E2* formation would seem to be a splicing related phenomenon. However, E2* formation does not require all the components necessary for in vitro splicing. E2* formation was supported by S100 extracts and mildly heat treated nuclear extracts, both of which were inactive in splicing. Retardation gel asssays and combined RNase T1 digestion and immunoprecipitation experiments indicated that large spliceosome-like complexes did not form on transcripts prepared with ATPaS. Furthermore, neither the absence of a functional 5' splice site nor polypyrimidine tract sequence prevented E2* formation, despite both these sequences being required for splicing. In addition, a nuclease activity has been identified in HeLa nuclear extract which is dependent on intact small nuclear RNAs U1 and U2. This activity may be involved in E2* formation.