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。MALAT1是一种丰富的、保守的、定位于致癌的核斑点 促进乳腺肿瘤进展和转移的lncRNA。MALAT1在低氧时被诱导，并且 调节低氧反应相关基因的前-mRNA选择性剪接(AS)。然而， MALAT1在低氧信号、肿瘤进展和血管生成中调控AS的分子机制 转移情况尚不清楚。全基因组RNA作图研究显示MALAT1与 转录活性基因及其前信使核糖核酸。此外，MALAT1还与 SR-剪接因子家族(SRSFs)和MALAT1表达失控的细胞在 SRSF水平和定位以及AS的异常。本提案的目的是界定 利用低氧反应研究MALAT1在SRSF介导的AS中的分子功能 模型系统。中心假设是，MALAT1通过在核斑点中富含SRSFs来调制 SRSFs与其目标前mRNAs和其他斑点驻留蛋白的结合。以Strong为指导 初步数据，这一假设将在以下具体目标进行检验：1)确定MALAT1 调节SRSF介导的选择性剪接(AS)。2)确定核斑点的重要性 MALAT1在mRNA加工中的富集性。在第一个目标中，PI将确定MALAT1如何调节 SRSF1(SRSF蛋白的原型成员)与其靶前mRNAs的结合和募集 缺氧性乳腺癌细胞。PI还将决定MALAT1在体内缺氧过程中的意义 使用肿瘤小鼠模型进行反应。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MALAT1参与1)斑点成分的空间组织， 2)调节斑点和AS附近基因的定位。这种方法在技术上是创新的， 因为它使用了最先进的细胞生物技术，包括超分辨率成像和基因 CRISPR/dCasRx介导的RNA拴系分析等方法。拟议的研究是 意义重大，因为破译MALAT1在调节基因表达中的作用将具有广泛的意义 在乳腺癌治疗背景下的翻译意义。最终，这些知识将为 未来利用MALAT1作为治疗癌症的新靶点的研究。

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.