Characterization of nuclear-retained RNA-mediated gene regulatory mechanisms

人类基因组编码大量的非蛋白质编码RNA（ncRNA）基因，包括数千个 长ncRNA（lncRNA）基因。MALAT 1是一种丰富的、保守的、核斑点定位的致癌基因， 促进乳腺肿瘤进展和转移的lncRNA。MALAT 1在缺氧期间被诱导，并且 调节参与缺氧反应的基因的前体mRNA选择性剪接（AS）。但 MALAT 1在缺氧信号传导、肿瘤进展和动脉粥样硬化过程中控制AS的分子机制 转移仍有待阐明。全基因组RNA作图研究表明，MALAT 1与 转录活性基因及其前体mRNA。此外，MALAT 1与几个成员的相互作用， SR家族剪接因子（SRSFs）和MALAT 1表达失调的细胞显示出在 SRSF水平和定位沿着与异常AS。本建议的目的是界定 以缺氧反应为实验手段，探讨MALAT 1在SRSF介导的AS中的分子功能， 模型系统核心假设是MALAT 1通过在核散斑中富集SRSFs， SRSF与其靶前mRNA和其他斑点驻留蛋白的结合。以强为导 初步数据，这一假设将在以下具体目标进行测试：1）确定如何MALAT 1 调节SRSF介导的可变剪接（AS）。2)确定核散斑的意义 MALAT 1在mRNA加工中的富集。在第一个目标中，PI将确定MALAT 1如何调节 SRSF 1（SRSF蛋白的原型成员）与其靶前体mRNA的结合和募集， 低氧乳腺癌细胞。PI还将确定MALAT 1在体内缺氧期间的意义 使用肿瘤小鼠模型的反应。在第二个目标下，PI通过使用超分辨率和实时成像 研究将确定MALAT 1参与，1）散斑成分的空间组织， （2）斑点和AS附近基因的调控定位。这种方法在技术上是创新的， 因为它采用了最先进的细胞生物学技术，包括超分辨率成像和遗传学， 方法，如CRISPR/dCasRx介导的RNA系链测定。拟议的研究是 重要的是，因为破译MALAT 1在调节基因表达中的作用将具有广泛的意义。 在乳腺癌治疗中的翻译意义。最终，这些知识将为 未来的研究利用MALAT 1作为一种新的治疗癌症的目标。

Human genome encodes a large number of non-protein coding RNA (ncRNA) genes, including thousands long ncRNA (lncRNA) genes. MALAT1 is an abundant, conserved and nuclear speckle localized oncogenic lncRNA that promotes breast tumor progression and metastasis. MALAT1 is induced during hypoxia, and regulates the pre-mRNA alternative splicing (AS) of genes involved in hypoxia response. However, the molecular mechanisms by which MALAT1 controls AS during hypoxia signaling, tumor progression and metastasis remain to be elucidated. Genome-wide RNA mapping studies reveal that MALAT1 interacts with transcriptionally active genes and their pre-mRNA. Further, MALAT1 interacts with several members of the SR-family of splicing factors (SRSFs), and cells with deregulated expression of MALAT1 show defects in SRSF levels and localization along with aberrant AS. The objective of the present proposal is to delineate the molecular function of MALAT1 in SRSF-mediated AS, by utilizing hypoxia response as an experimental model system. The central hypothesis is that MALAT1 by enriching SRSFs in nuclear speckles, modulates the binding of SRSFs with their target pre-mRNAs and other speckle resident proteins. Guided by strong preliminary data, this hypothesis will be tested in the following specific aims: 1) Determine how MALAT1 regulates SRSF-mediated alternative splicing (AS). 2) Determine the significance of nuclear speckle enrichment of MALAT1 in mRNA processing. In the first aim, PI will determine how MALAT1 regulates the binding and recruitment of SRSF1 (a prototypical member of SRSF proteins) to their target pre-mRNAs in hypoxic breast cancer cells. PI will also determine the significance of MALAT1 during in vivo hypoxia response using tumor mouse models. Under the second aim, PI by using super-resolution and live imaging studies will determine the involvement of MALAT1 in the, 1) spatial organization of speckle components, and 2) regulated localization of genes near speckles and AS. The approach is technically innovative, because it employs state of the art cell biological techniques, including super-resolution imaging and genetic approaches such as CRISPR/dCasRx-mediated RNA tethering assays. The proposed research is significant because deciphering the role of MALAT1 in regulating the expression of genes will have broad translational significance in the context of breast cancer treatment. Ultimately, this knowledge will pave way to future studies utilizing MALAT1 as a novel therapeutic target against cancer.