Biology of terminal R-loops in splicing factor mutant cancers

这个重新提交的R 03申请旨在确定剪接因子（SF）中常见的复发性突变是如何发生的。 导致染色质可及性和表观遗传景观的变化，从而促进肿瘤发生。 SF中的复发性突变在多种不相关的癌症类型中普遍存在，包括急性髓系 白血病、慢性淋巴细胞白血病、肺癌和黑色素瘤。在数以百计的剪接因子中， 只有4种常见突变：SF 3B 1、SRSF 2、U2 AF 1和ZRSR 2。SF突变是相互排斥的， 非同义词，表明它们作为具有新形态功能的肿瘤驱动因子的作用。他们的既定角色 在剪接催化作用中的研究已经导致了这样的假设，即肿瘤抑制基因或癌基因的选择性剪接是 驱动突变细胞克隆进化的主要机制。虽然选择性剪接事件是 在RNA-seq数据集中可以证明，这种变化是相当温和的，并限于特定的突变。 亚型这使得选择性剪接在肿瘤发生中的直接作用受到质疑。最近的研究 研究了肿瘤发生的替代分子机制，包括过量的R环， 突变体SF表达。R-环是由两个DNA分子和一个RNA分子组成的3链结构， 转录。R环是染色质状态的关键调节器，并且当不受抑制时可导致基因组突变。 不稳定这些R环也导致S期停滞，并且可以通过RNAseH的过表达来挽救。 新出现的证据指出剪接体机制和信使RNA之间的密切协调 加工，包括其3'端切割和终止。因此，我们假设，终端R环出现， mRNA加工缺陷的结果在这个建议的第一个目标，我们将确定转录动力学 通过新生RNA的代谢标记（瞬时转录测序）在SF 3B 1和U2 AF 1突变中 用Timelapse化学或TT-TL-seq）。RNA聚合酶II相对于R环的位置和停滞 DNA复制叉将通过邻位连接试验测定。第二个目标将探讨如何 表观遗传修饰物调节这种病理性R环的形成和分解。我们的初步结果 使用短发夹RNA文库揭示了组蛋白去乙酰化酶途径在这种调节中的作用;因此，我们 将探讨HDAC和R环之间的关系。最后，我们将确定染色质 剪接因子突变型急性髓性白血病患者样本的可及性，因为R环和开放 染色质高度相关。鉴于R 03机制的范围有限，该建议主要是 专注于连接异常RNA加工和R环形成的生物化学研究。如果成功，它将 形成的基础上，全面调查的机制，肿瘤赋予SF 利用适当的体内模型进行突变。

This resubmitted R03 application seeks to determine how recurrent mutations common in splicing factors (SF) lead to changes in chromatin accessibility and epigenetic landscape, thereby contributing to oncogenesis. Recurrent mutations in SF are prevalent across multiple unrelated cancer types including acute myeloid leukemia, chronic lymphocytic leukemia, lung cancer and melanoma. Among the hundreds of splicing factors, only 4 are commonly mutated: SF3B1, SRSF2, U2AF1 and ZRSR2. SF mutations are mutually exclusive and non-synonymous, suggesting their role as tumor drivers with neomorphic function. Their well-established roles in splicing catalysis have led to the presumption that alternate splicing of tumor suppressors or oncogenes is the primary mechanism driving clonal evolution of mutant cells. While alternate splicing events are demonstrable in RNA-seq datasets, such changes are quite modest and restricted to specific mutational subtypes. This makes a direct role of alternative splicing in oncogenesis questionable. Recent studies have looked at alternate molecular mechanisms of oncogenesis including excess R-loops that arise in response to mutant SF expression. R-loops are 3 stranded structures of two DNA and one RNA molecules formed during transcription. R-loops are critical regulators of chromatin states, and when unchecked can lead to genome instability. These R-loops also cause S-phase arrest and can be rescued by the over-expression of RNAseH. Emerging evidence points to the close coordination between spliceosomal machinery and messenger RNA processing, including its 3' end cleavage and termination. We hence hypothesize that terminal R-loops arise from defective mRNA processing. In the first aim of this proposal, we will determine dynamics of transcription in SF3B1 and U2AF1 mutations through metabolic labeling of nascent RNA (transient transcript sequencing with Timelapse chemistry or TT-TL-seq). Location of RNA Polymerase II in relation to R-loops and stalled DNA replication forks will be determined through proximity ligation assay. The second aim will explore how epigenetic modifiers regulate the formation and resolution of such pathologic R-loops. Our preliminary results using a short hairpin RNA library reveals the role of histone deacetylase pathway in this regulation; hence we will explore the relation between HDAC and R-loops in this aim. Finally, we will determine chromatin accessibility in splicing factor mutant acute myeloid leukemia patient samples, since R-loops and open chromatin are highly correlated. Given the limited scope of the R03 mechanism, the proposal is primarily focused on biochemical studies linking aberrant RNA processing and R-loop formation. If successful, it will form the basis of comprehensive investigations into the mechanisms of oncogenesis conferred by SF mutations utilizing appropriate in vivo models.