Pacheco Teresa Raquel, Moita Luís Ferreira, Gomes Anita Quintal, Hacohen Nir, Carmo-Fonseca Maria
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Mol Biol Cell. 2006 Oct;17(10):4187-99. doi: 10.1091/mbc.e06-01-0036. Epub 2006 Jul 19.
U2AF is a heterodimeric splicing factor composed of a large (U2AF65) and a small (U2AF35) subunit. In humans, alternative splicing generates two U2AF35 variants, U2AF35a and U2AF35b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF35a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF35a affected the expression level of approximately 500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF35a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF35a or U2AF35b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF35a is essential for HeLa cell division and suggest a novel role for both U2AF35 protein isoforms as regulators of alternative splicing of a specific subset of genes.
U2AF是一种异源二聚体剪接因子，由一个大亚基（U2AF65）和一个小亚基（U2AF35）组成。在人类中，可变剪接产生两种U2AF35变体，即U2AF35a和U2AF35b。在此，我们利用RNA干扰特异性地消除了HeLa细胞中每种异构体的表达。我们的结果表明，主要异构体U2AF35a的敲低降低了细胞活力并损害了有丝分裂进程，导致细胞在前中期积累。微阵列分析显示，U2AF35a的敲低影响了约500种mRNA的表达水平，其中超过90%相对于对照表达不足。在U2AF35a缺失细胞中表达不足的mRNA中，我们鉴定出一个必需的细胞周期基因Cdc27，其未剪接RNA与剪接RNA的比例增加，蛋白质水平显著降低。此外，我们表明，U2AF35a或U2AF35b的缺失改变了Cdc25B和Cdc25C转录本可变剪接异构体的比例。综上所述，我们的结果表明U2AF35a对HeLa细胞分裂至关重要，并提示两种U2AF35蛋白异构体作为特定基因子集可变剪接的调节因子具有新的作用。
