Proteomic study identifies Aurora-A mediated regulation of alternative splicing through multiple splicing factors. 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. Overall design: 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）是癌症治疗的极具吸引力的靶点，但其抑制作用亦会伴随毒性副作用。为更精细地解析极光激酶A的功能，我们采用鸟枪法蛋白质组学（shotgun proteomics）鉴定出407个特异性蛋白互作伴侣，其中包含多种剪接因子。研究发现，极光激酶定位于核斑（nuclear speckles）——剪接蛋白的储存库，这一结果支持其在可变剪接中发挥作用的推测。极光激酶在体外与体内均能与剪接因子相互作用并使其磷酸化，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。与之相一致的是，极光激酶抑制会显著影响505个基因的可变剪接，RNA基序分析表明，极光激酶互作的剪接因子存在富集现象。此外，我们还观察到极光激酶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。一个有趣的案例为CLK1外显子4，其似乎通过SRSF3受极光激酶A调控。综上，本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。
实验设计概述：我们在HEK293T细胞中分别通过RNA干扰（RNAi）技术抑制极光激酶A（处理24小时）或敲低内源性剪接因子（处理48小时），并以非靶向小干扰RNA（siRNAs）作为对照，随后开展RNA测序（RNA sequencing），以探究极光激酶A与关键剪接调控因子之间的功能关联。