Dagher S F, Wang J L, Patterson R J
Genetics Program, Michigan State University, East Lansing 48824.
Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1213-7. doi: 10.1073/pnas.92.4.1213.
Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.
半乳糖凝集素-3（分子量约为35,000）是一种半乳糖/乳糖特异性凝集素，在许多动物细胞中与核糖核蛋白复合物相关联。通过使用32P标记的MINX作为前体mRNA底物，进行了无细胞剪接试验，以研究HeLa细胞核提取物在RNA加工过程中对半乳糖凝集素-3的需求。添加与半乳糖凝集素-3具有高亲和力的糖类会抑制剪接试验中的产物形成，而添加不与该凝集素结合的碳水化合物则不会抑制产物形成。通过乳糖琼脂糖柱亲和吸附去除半乳糖凝集素-3的细胞核提取物缺乏剪接活性。在对照纤维二糖琼脂糖上进行平行吸附的提取物保留了剪接活性。去除半乳糖凝集素-3的提取物的活性可以通过添加纯化的重组半乳糖凝集素-3来恢复，而添加其他具有相似糖类结合特异性的凝集素（大豆凝集素）或具有不同特异性的凝集素（麦胚凝集素）则不能恢复剪接活性。剪接复合物的形成对半乳糖凝集素-3的去除和重建也很敏感。总之，这些结果确定了前体mRNA剪接对半乳糖凝集素-3的需求，并将其鉴定为一种剪接因子。
