Department of Biochemistry and Molecular Biology, Michigan State University.
Department of Microbiology and Molecular Genetics, Michigan State University.
J Vis Exp. 2020 Dec 9(166). doi: 10.3791/61990.
Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.
经典的耗尽-再构成实验表明半乳糖凝集素-3 是核提取物中必需的剪接因子。本文探讨了半乳糖凝集素-3 掺入剪接途径的机制。在 12%-32%甘油梯度上沉淀 HeLa 细胞核提取物，可得到富含内源性~10S 颗粒的级分，该颗粒含有半乳糖凝集素-3 和 U1 snRNP。我们现在描述了一种用抗半乳糖凝集素-3 抗体共价偶联琼脂糖珠上捕获的半乳糖凝集素-3-U1 snRNP 颗粒来耗尽核提取物中 U1 snRNP 同时丧失剪接活性的方案。在耗尽 U1 的提取物中，U1 耗尽的提取物中的剪接活性可以通过半乳糖凝集素-3-U1 snRNP-前 mRNA 三元复合物来重建，该复合物是一种功能性 E 复合物，导致剪接反应的中间产物和产物，并且半乳糖凝集素-3 通过与 U1 snRNP 结合进入剪接途径。使用在珠子上通过复合物亲和或免疫选择来重建特定剪接因子耗尽的提取物中的剪接活性的方案可能普遍适用于其他系统。
