Proteomic study identifies Aurora-A mediated regulation of alternative splicing through multiple splicing factors

The cell cycle regulator Aurora-A kinase presents an attractive target for cancer therapies, though its inhibition is also associated with toxic side effects. To gain a more nuanced understanding of Aurora-A function, we applied shotgun proteomics to identify 407 specific protein partners, including several splicing factors. Supporting a role in alternative splicing, we found that Aurora-A localizes to nuclear speckles, the storehouse of splicing proteins. Aurora-A interacts with and phosphorylates splicing factors both in vitro and in vivo, suggesting that it regulates alternative splicing by modulating the activity of these splicing factors. Consistently, Aurora-A inhibition significantly impacts the alternative splicing of 505 genes, with RNA motif analysis revealing an enrichment for Aurora-A interacting splicing factors. Additionally, we observed a significant positive correlation between the splicing events regulated by Aurora-A and those modulated by its interacting splicing factors. An interesting example is represented by CLK1 exon 4, which appears to be regulated by Aurora-A through SRSF3. Collectively, our findings highlight a broad role of Aurora-A in the regulation of alternative splicing. RNA sequencing was performed in HEK293T cells either with Aurora-A inhibition (24 hours) or depleted of endogenous splicing factors (48 hours) using RNAi (non-targeting siRNAs were used as control) to explore a functional link between Aurora-A and key splicing regulators.

细胞周期调控因子极光激酶A（Aurora-A kinase）是癌症治疗领域极具吸引力的靶点，但其抑制作用也会伴随毒性副作用。为更细致深入地解析极光激酶A的功能，我们采用鸟枪法蛋白质组学（shotgun proteomics）鉴定出407种特异性蛋白质互作伙伴，其中包含多种剪接因子。为佐证其在可变剪接中的调控作用，我们发现极光激酶A定位于核斑（nuclear speckles）——剪接蛋白的储存库。极光激酶A在体外与体内均可与剪接因子结合并使其磷酸化，这表明其通过调控此类剪接因子的活性来参与可变剪接调控。与之相符的是，极光激酶A的抑制会显著影响505个基因的可变剪接模式，RNA基序分析显示，该激酶的互作剪接因子存在富集现象。此外，我们还观察到极光激酶A调控的剪接事件与其互作剪接因子所调控的剪接事件之间存在显著正相关。一个典型案例为CLK1外显子4，其似乎可通过SRSF3受极光激酶A调控。综上，我们的研究结果揭示了极光激酶A在可变剪接调控中的广泛作用。为探究极光激酶A与关键剪接调控因子之间的功能关联，我们对HEK293T细胞开展了RNA测序实验：一组采用极光激酶A抑制剂处理24小时，另一组通过RNA干扰（RNAi）敲除内源性剪接因子（以非靶向siRNA作为对照，处理时长为48小时）。