Zillmann M, Zapp M L, Berget S M
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Mol Cell Biol. 1988 Feb;8(2):814-21. doi: 10.1128/mcb.8.2.814-821.1988.
Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.
在体外剪接提取物中孵育期间，通过一种新的核糖核蛋白（RNP）凝胶电泳系统监测剪接前体RNA组装成核糖核蛋白颗粒（RNP）复合物的过程。我们的系统观察到的组装时间模式与通过其他凝胶和梯度方法获得的模式相同。然而，与其他系统获得的结果不同，我们观察到形成特定复合物以及在凝胶分离的复合物中保留U1小核核糖核蛋白（snRNP）需要U1 snRNP和5'剪接连接序列。单内含子底物RNA迅速组装成迁移缓慢的复合物。第一个特定复合物（A）在孵育一分钟内出现，形成需要ATP、5'和3'前体RNA共有序列以及完整的U1和U2 RNA。孵育15分钟后出现了含有前体RNA的第二个复合物（B）。套索状外显子2和外显子1中间体首先出现在这个复合物中，从操作上定义它为活性剪接体。复合物B的出现需要U4 RNA。释放的套索首先出现在迁移率中等的复合物（A'）中，随后出现在迁移迅速的弥散复合物中。仅在迁移迅速的复合物中观察到连接产物RNA。U1 snRNP被检测为凝胶分离复合物的成分。在凝胶上样条件下以及从凝胶分离的复合物中，A和B复合物中的放射性标记RNA被U1特异性抗体免疫沉淀。因此，在凝胶电泳期间，RNP抗原仍与组装好的复合物相关联。此外，在存在互补寡核苷酸的情况下，凝胶分离的A和B复合物中的5'剪接连接序列对RNase H切割不可接近。因此，在我们的凝胶条件下，结合5'剪接连接的核因子也仍与5'剪接连接相关联。
