Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2022;2442:713-726. doi: 10.1007/978-1-0716-2055-7_38.
Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.
通过甘油梯度离心对 HeLa 细胞核提取物进行分级分离，将内源性富含尿嘧啶的小核核糖核蛋白复合物（U snRNP）分离成从 7S 到大于 60S 的多个颗粒。沉降在 10S 的复合物含有单个 U snRNP（U1 snRNP）和半乳糖凝集素-3。将针对半乳糖凝集素-3 的特异性抗体添加到包含这些 10S 复合物的级分中，可共沉淀 U1 snRNP，表明一部分 U1 snRNP 与该半乳糖凝集素相关。通过耗尽-重建研究表明，半乳糖凝集素-3 是一个完整的剪接成分，参与剪接体组装和剪接活性。剪接体组装的第一步是 U1 snRNP 与前信使 RNA 底物的 5'剪接位点结合。在剪接提取物中发现 U1 snRNP 和半乳糖凝集素-3 相关，这暗示该复合物为半乳糖凝集素-3 进入剪接途径提供了一个潜在的入口。从甘油梯度的 10S 区域免疫选择 U1 snRNP-半乳糖凝集素-3 复合物添加到 U1 耗尽的核提取物中，可引发剪接反应，形成剪接中间体和成熟的 mRNA。本章描述了这些实验的材料和方法，这些实验证明 U1 snRNP-半乳糖凝集素-3 复合物在 U1 耗尽的核提取物中引发剪接反应。
