Günzl A, Cross M, Palfi Z, Bindereif A
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Federal Republic of Germany.
J Biol Chem. 1993 Jun 25;268(18):13336-43.
trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.
锥虫中的反式剪接需要U2和U4/U6小核（sn）核糖核蛋白发挥作用。我们利用针对U2 snRNP蛋白的特异性抗体以及U2缺失衍生物和人-锥虫杂交RNA的体外重组试验，分析了布氏锥虫U2 snRNP的蛋白结合和组装情况。U2特异性40 kDa蛋白和常见蛋白的稳定结合仅需要3'-末端结构域（茎环IIb、单链区域和茎环IV），其中环IV提供关键的序列决定因素；茎环IV足以结合40 kDa蛋白，但不足以结合常见蛋白；令人惊讶的是，茎环IIb和IV之间“类Sm”单链区域的序列对于蛋白结合并非必需。我们的突变分析进一步表明，常见蛋白和特异性蛋白之间的相互作用在稳定核心复合物的组装中起重要作用。最后，部分组装的U2 RNP复合物可被鉴定为U2 snRNP组装的动力学中间体。我们提出了反式剪接体U2 snRNP的结构域结构和组装模型，该模型在几个方面与顺式剪接体U2 snRNP不同；这些差异可能与锥虫U2 snRNP的反式剪接特异性功能有关。
