Moore C L, Skolnik-David H, Sharp P A
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Mol Cell Biol. 1988 Jan;8(1):226-33. doi: 10.1128/mcb.8.1.226-233.1988.
Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.
含有腺病毒L3聚腺苷酸化位点的前体RNA在30℃下与HeLa细胞核提取物一起温育时会组装成一个50S复合物。50S复合物形成的辅因子和序列要求与体外聚腺苷酸化反应相似。该复合物的组装需要ATP，但不依赖于多聚腺苷酸尾的合成。此外，在聚腺苷酸化位点上游的AAUAAA六核苷酸已突变为AAGAAA的底物RNA上，或在该位点下游+5至+48个核苷酸之间的序列已被去除的RNA上，不会形成50S复合物。这些突变也会阻止底物RNA的体外加工。动力学研究表明，50S复合物是聚腺苷酸化反应的中间体。它在反应的早期形成，在后期既包含多聚腺苷酸尾化的RNA，也包含未反应的前体。U型小核核糖核蛋白颗粒是50S复合物的组成成分，这通过用针对这些小核RNA三甲基帽的抗血清进行免疫沉淀得以证明。
