Deirdre A, Scadden J, Smith C W
Department of Biochemistry, University of Cambridge, UK.
EMBO J. 1995 Jul 3;14(13):3236-46. doi: 10.1002/j.1460-2075.1995.tb07326.x.
Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome. We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron. As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of terminal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing. Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing. Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired. Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.
核前体mRNA剪接具有与II类自剪接内含子所采用的机制基本相似的两步机制。据信，核前体mRNA剪接涉及形成活性剪接体催化核心的RNA-RNA相互作用网络。我们在此展示了哺乳动物内含子的第一个和最后一个鸟苷残基之间的非沃森-克里克相互作用。与酿酒酵母一样，分别用A和C取代5'和3'剪接位点处的保守鸟苷，可特异性抑制由单个突变导致的第二步剪接缺陷。没有其他末端核苷酸组合能够恢复剪接。我们还提供了独立证据，证明在剪接的第二步中，共有剪接位点的其他核苷酸之间存在间接相互作用。5'剪接位点+3位置的核苷酸取代会影响3'剪接位点处紧密间隔的AG二核苷酸之间的竞争，尽管这种相互作用不是通过直接的差异碱基配对。最后，我们表明，在pre-mRNA中用次黄嘌呤完全取代鸟苷残基对剪接的第二步只有适度影响，尽管早期的剪接体组装步骤会受到损害。因此，可以对末端残基之间非沃森-克里克相互作用的精确构型进行预测。
