Examination of the role of galectins in pre-mRNA splicing.
Examination of the role of galectins in pre-mRNA splicing.
复制标题
检查半乳糖凝集素在前 mRNA 剪接中的作用。
DOI：
10.1007/978-1-4939-1396-1_28
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发表时间：
2015
影响因子：
--
通讯作者：
John L. Wang
中科院分区：
文献类型：
--
作者：
R. Patterson;Kevin C. Haudek;P. G. Voss;John L. Wang
已经积累了几条证据表明半乳糖凝集素-1和半乳糖凝集素-3是参与前体mRNA核剪接的许多蛋白质中的两种。首先，能够在无细胞测定中进行前mRNA剪接的核提取物含有两种半乳糖凝集素。其次，使用乳糖亲和层析或抗体免疫吸附从核提取物中耗尽半乳糖凝集素，导致剪接活性的伴随损失。第三，将半乳糖凝集素-1或半乳糖凝集素-3添加到半乳糖凝集素耗尽的提取物中重建剪接活性。第四，添加以高亲和力结合半乳糖凝集素-1和半乳糖凝集素-3的寡核苷酸（例如，乳糖或硫代二半乳糖苷）加入核提取物导致剪接的抑制，而平行加入不与半乳糖凝集素结合的β-半乳糖苷（例如，纤维二糖）不能产生相同的效果。最后，当剪接反应通过针对半乳糖凝集素-1的抗体进行免疫沉淀时，对应于起始前mRNA底物、成熟mRNA产物和剪接反应中间体的放射性标记的RNA种类与半乳糖凝集素共沉淀。用抗半乳糖凝集素-3的抗体也获得了类似的结果。本章介绍了我们的研究中使用的两个关键检测：一个报告的剪接活性，通过观察在变性凝胶上的产物形成;其他报告的剪接体组装使用非变性或天然凝胶的中间体。
Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.
在活细胞中探测核糖核蛋白颗粒。
DOI：
10.1073/pnas.78.4.2208
发表时间：
1981
影响因子：
11.1
作者：
Mayrand,S;Pederson,T
通讯作者：
Pederson,T
影响因子：
13.8
作者：
Hoskins, Aaron A.;Moore, Melissa J.
通讯作者：
Moore, Melissa J.