PO.MCB07.03 · 分子与细胞生物学
剪接因子突变型癌症中共有剪接异常的统一机制
A unifying mechanism for shared splicing aberrations in splicing factor mutant cancers
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
剪接因子（SF）如SF3B1、U2AF1和SRSF2中的癌症相关突变会产生公认的、特定于各突变亚型的顺式作用剪接位点选择改变。然而，在这些遗传学上不同的突变组之间，可变剪接（AS）缺陷存在大量重叠，此现象一直未得到解释。我们推测这种趋同反映了一种共有的反式调控机制。为回答此问题，我们对全转录组进行了分析，利用一个包含395例SF突变克隆性髓系疾病患者和64名健康供者的队列，刻画SF突变状态下的AS程序。虽然大多数AS改变是突变特异性的，但我们鉴定出一个稳健的共有事件子集，其显著富集于内含子保留（RI）特征。这些RI缺陷是双向的（RI既增加又减少），但在SF3B1、U2AF1和SRSF2突变体之间却惊人地一致。鉴于RNA结合蛋白（RBP）是AS的主要调控者，我们将这些SF突变型RI与356个单独RBP缺失细胞（ENCODE）中的RI进行比较。SF突变型RI与SRSF1功能缺失所导致的RI高度相似。磷酸化蛋白质组学揭示，SF突变细胞中SRSF1的RS结构域发生低磷酸化，损害其剪接活性。这种降低源于AMPKalpha-AKT信号平衡的转变，后者抑制了AKT-SRPK1-SRSF1磷酸化轴。我们接着将这一信号失衡追溯至一个上游触发因素：R环相关的转录应激，它激活DNA损伤反应（DDR）。DDR激活增强AMPKalpha信号，同时削弱AKT活性，从而降低SRPK1介导的SRSF1磷酸化。药理学激活DDR再现了SRSF1的低磷酸化，降低了AKT/SRPK1活性，并诱导出类似SF突变细胞中的RI缺陷。相反，解除DDR信号可恢复AKT/SRPK1活性、使SRSF1磷酸化正常化并纠正RI异常。这些结果在患者来源的SF突变克隆性髓系细胞中得到证实——通过过表达RNaseH逆转R环，并用CFU实验量化其对集落形成的影响。总之，我们的发现表明，除了各自不同的顺式作用剪接位点改变外，SF突变型癌症还共有一种应激驱动的反式作用剪接程序，该程序通过DDR介导的SRSF1活性重塑而协调。这一统一机制在多样化的克隆状态中将复制应激、激酶信号和RNA加工联系起来，并突出了具有治疗潜力的新型可操作节点（DDR、AMPK/AKT平衡以及SRPK1-SRSF1偶联）。
查看英文原文 English abstract
Cancer-associated mutations in splicing factors (SFs) such as SF3B1, U2AF1, and SRSF2 generate well-recognized cis-acting alterations in splice site choice, specific to each mutational subtype. However, there is substantial overlap in alternative splicing (AS) defects across these genetically distinct mutation groups which has remained unexplained. We hypothesized that this convergence reflects a shared trans-regulatory mechanism. To answer this, we profiled global transcriptomes to delineate AS programs across SF-mutant states using a cohort of 395 patients with SF-mutant clonal myeloid disorders and 64 healthy donors. While the majority of AS alterations were mutation-specific, we identified a robust subset of shared events, strongly enriched for retained intron (RI) signatures. These RI defects were bidirectional (both increase and decrease in RI) yet strikingly concordant across SF3B1, U2AF1, and SRSF2 mutants. Given that RNA binding proteins (RBPs) are the primary regulators of AS, we compared these SF-mutant RI to those in cells with loss of 356 individual RBPs (ENCODE). SF-mutant RI closely mirrored RI in resulting from SRSF1 loss of function. Phosphoproteomics revealed that SF-mutant cells have hypophosphorylation of RS domains in SRSF1, impairing its splicing activity. This reduction stemmed from a shift in the AMPKalpha-AKT signaling balance, which suppressed the AKT-SRPK1-SRSF1 phosphorylation axis. We next traced this signaling imbalance to an upstream trigger: R-loop-associated transcriptional stress, which activates a DNA damage response (DDR). DDR activation enhanced AMPKalpha signaling while diminishing AKT activity, thereby reducing SRPK1-mediated phosphorylation of SRSF1. Pharmacologic DDR activation recapitulated SRSF1 hypophosphorylation, decreased AKT/SRPK1 activity, and induced RI defects resembling those in SF-mutant cells. Conversely, relief of DDR signaling restored AKT/SRPK1 activity, normalized SRSF1 phosphorylation, and corrected RI abnormalities. These results were confirmed in SF-mutant clonal myeloid cells from patients using RNAseH over-expression to reverse R-loops and quantifying effect on colony formation by CFU assays. Together, our findings demonstrate that, beyond distinct cis-acting splice site changes, SF-mutant cancers share a stress-driven, trans-acting splicing program coordinated through DDR-mediated rewiring of SRSF1 activity. This unified mechanism links replication stress, kinase signaling, and RNA processing across diverse clonal states, and highlights novel actionable nodes (DDR, AMPK/AKT balance, and SRPK1-SRSF1 coupling) with therapeutic potential.
利益披露 Disclosure
R. Roy, None..
P. C. Boddu, None..
M. M. Pillai, None.