Tarn W Y
Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan.
Biochimie. 1996;78(11-12):1057-65. doi: 10.1016/s0300-9084(97)86730-2.
Genetic evidence in yeast has revealed that a non-Watson-Crick base-pairing interaction between terminal guanosine residues of the intron is required for the second step of pre-mRNA splicing. To explore the likely configuration of the interaction between the terminal guanosines of the intron, inosine was uniformly incorporated into an adenovirus pre-mRNA substrate (Ade) to replace guanosine residues. Splicing of the inosine-containing Ade pre-mRNA was completely inhibited. Psoralen cross-linking reveals that the association of U1 and U2 snRNPs with the intron was impaired. To eliminate the deleterious effects caused by complete inosine replacement, guanosine residues at the splice site(s) of the Ade pre-mRNA were substituted by inosine. Such pre-mRNA substrates were obtained by ligation of two or three RNA fragments; the 3' piece was primed with inosine or mono-phosphate inosine. Splicing of the Ade pre-mRNA containing an inosine residue at the 5' or the 3' splice site, or at both sites proceeds normally. Thus, the functions of the terminal guanosine residues of the intron in splicing can be replaced by inosine. This result supports the previous notion that an N1-carbonyl symmetric interaction likely occurs between the intron terminal residues during pre-mRNA splicing.
酵母中的遗传证据表明，前体mRNA剪接的第二步需要内含子末端鸟苷残基之间形成非沃森-克里克碱基配对相互作用。为了探究内含子末端鸟苷之间相互作用的可能构型，将肌苷均匀掺入腺病毒前体mRNA底物（Ade）中以取代鸟苷残基。含肌苷的Ade前体mRNA的剪接被完全抑制。补骨脂素交联显示U1和U2 snRNP与内含子的结合受损。为了消除完全由肌苷取代所造成的有害影响，将Ade前体mRNA剪接位点处的鸟苷残基替换为肌苷。此类前体mRNA底物通过连接两个或三个RNA片段获得；3'片段用肌苷或肌苷单磷酸进行引物延伸。在5'或3'剪接位点或两个位点均含有一个肌苷残基的Ade前体mRNA的剪接正常进行。因此，内含子末端鸟苷残基在剪接中的功能可被肌苷取代。这一结果支持了先前的观点，即在mRNA前体剪接过程中，内含子末端残基之间可能发生N1-羰基对称相互作用。
