Investigations of Consequences of U2AF1 Mutations in MDS

描述(申请人提供)：体细胞突变、染色体缺陷和表观遗传学改变是骨髓增生异常综合征(MDS)的关键致病缺陷。分子技术的最新进展导致了新的一类复发病变的发现，并识别了与特定病理形态特征相关的肿瘤发生或突变的新的分子途径。这项建议集中在一类新发现的影响剪接体基因的新突变上，其中包括U2AF1。在MDS和AML中，影响该基因2个锌指结构域的高度重复的杂合性错义突变在MDS和AML中很常见，它们预示着进展加速和生存不良。由于剪接体突变似乎在某些形式的MDS中特别频繁，这种疾病将作为研究剪接体缺陷介导致癌效应的机制的模型。我们的建议是基于这样的假设，即由于体细胞突变导致剪接体基因的缺陷导致特定类型的肿瘤抑制基因(TSG)的错误剪接，从而最终导致类似于直接损伤这些TSG所产生的致病后果。因此，剪接体突变可能是其他基因组缺陷的表型后果。在分子水平上，U2AF1突变通过对特定剪接位点序列的差异排除而导致“功能改变”，从而导致特定的错接模式的产生。在这个项目中，我们将研究反复出现的错误剪接模式和结构与功能的关系 MDS中U2AF1突变导致的剪接缺陷，并识别受错误剪接影响的外显子。我们将确定在患者中观察到的剪接功能障碍和异常剪接模式是否可以在工程模型细胞系中重现。我们还将比较纯化的重组野生型和突变型U2AF的RNA结合特异性以及对错配目标基因体外剪接的影响。此外，我们将通过引入带有突变体结合位点的诱骗RNA来恢复细胞中的正常剪接，并研究抑制PP1/PP2磷酸酶是否可以改善剪接体功能。最后，我们将分析U2AF1突变的临床后果。突变相关的表型和结果将与那些没有U2AF1突变但下游基因单倍体表达/亚型功能不足的患者进行比较，这些下游基因被发现受到U2AF1缺陷的影响。

DESCRIPTION (provided by applicant): Somatic mutations, chromosomal defects and epigenetic changes constitute the key pathogenic defects in myelodysplastic syndrome (MDS). Recent scientific advances in molecular technologies have led to the discovery of new classes of recurrent lesions and the identification of novel molecular pathways of oncogenesis or mutations associated with specific pathomorphologic features. This proposal focuses on a newly discovered novel class of mutations affecting spliceosomal genes and among them U2AF1. Highly recurrent, heterozygous missense mutations affecting 2 zinc finger domains in this gene are frequent in MDS and AML where they are prognostic for accelerated progression and poor survival. Because spliceosomal mutations appear to be particularly frequent in certain forms of MDS, this disease will serve as a model for investigation of mechanisms by which spliceosomal defects mediate oncogenic effects. Our proposal is based on the hypothesis that defects in spliceosomal genes due to somatic mutations lead to specific types of mis- splicing of distinct combinations of tumor suppressor genes (TSG) and therefore ultimately result in pathogenetic consequences similar to those produced by direct lesions to these TSG. Hence, spliceosomal mutations may phenocopy consequences of other genomic defects. On the molecular level, U2AF1 mutations result in "change of function" through differential exclusion of specific splice site sequences and thereby result in creation of specific mis-splicing patterns. In this project, w will investigate the recurrent mis-splicing patterns and the structure-function relationship of the splicing defects due to U2AF1 mutations in MDS and identify exons affected by mis-splicing. We will determine whether splicing dysfunction and the aberrant splicing patterns observed in patients can be recapitulated in engineered model cell lines. We will also compare the RNA binding specificities of purified recombinant wild type and mutant U2AF as well as the effects on in vitro splicing of mispliced target genes. Furthermore, we will restore normal splicing in cells y introducing decoy RNAs with binding sites for the mutant and investigate whether the inhibition of PP1/PP2 phosphatases can improve spliceosomal function. Finally, we will analyze the clinical consequences of U2AF1 mutations. Mutation-associated phenotypes and outcomes will be compared to those seen in patients without U2AF1 mutations but with the haploinsufficient expression/hypomorphic function of downstream genes found to be otherwise affected in by U2AF1 defects.