Gaytan-Cervantes Javier, Gonzalez-Torres Carolina, Maldonado Vilma, Zampedri Cecilia, Ceballos-Cancino Gisela, Melendez-Zajgla Jorge
From the Laboratories of Functional Genomics and.
Epigenetics, National Institute of Genomic Medicine (INMEGEN), 14610 Mexico City, Mexico.
J Biol Chem. 2017 Aug 18;292(33):13745-13757. doi: 10.1074/jbc.M117.800318. Epub 2017 Jun 27.
Messenger RNA alternative splicing (AS) regulates the expression of a variety of genes involved in both physiological and pathological processes. AS of the anti-apoptotic and proliferation-associated survivin (BIRC5) gene generates six isoforms, which regulate key aspects of cancer initiation and progression. One of the isoforms is DEx3, in which the exclusion of exon 3 generates a unique carboxyl terminus with specific anti-apoptotic functions. This isoform is highly expressed in advanced stages of breast and cervical tumors. Therefore, understanding the mechanisms that regulate survivin DEx3 mRNA AS is clearly important. To this end, we designed a minigene (M), and in combination with a series of deletions and site-directed mutations, we determined that the first 22 bp of exon 3 contain -acting elements that enhance the exclusion of exon 3 to generate the survivin DEx3 mRNA isoform. Furthermore, using pulldown assays, we discovered that Sam68 is a possible acting factor that binds to this region and regulates exon 3 splicing. This result was corroborated using a cell line in which the Sam68 binding site in the gene was mutated with the CRISPR/Cas system. This work provides the first clues regarding the regulation of survivin DEx3 mRNA splicing.
信使核糖核酸可变剪接（AS）调控参与生理和病理过程的多种基因的表达。抗凋亡和增殖相关的生存素（BIRC5）基因的可变剪接产生六种异构体，这些异构体调控癌症发生和进展的关键方面。其中一种异构体是DEx3，外显子3的缺失产生具有特定抗凋亡功能的独特羧基末端。这种异构体在乳腺癌和宫颈癌的晚期高度表达。因此，了解调控生存素DEx3信使核糖核酸可变剪接的机制显然很重要。为此，我们设计了一个小基因（M），并结合一系列缺失和定点突变，我们确定外显子3的前22个碱基对包含增强外显子3缺失以产生生存素DEx3信使核糖核酸异构体的顺式作用元件。此外，通过下拉实验，我们发现Sam68是一个可能与该区域结合并调控外显子3剪接的作用因子。使用CRISPR/Cas系统对该基因中Sam68结合位点进行突变的细胞系证实了这一结果。这项工作为生存素DEx3信使核糖核酸剪接的调控提供了首个线索。
