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、保守、定位于核斑点的lncRNA。 这会促进乳腺肿瘤的转移。MALAT1在缺氧过程中被诱导了几倍，并影响了 低氧反应调控基因的前mRNA选择性剪接(AS)。然而，分子 MALAT1在低氧信号转导过程中控制AS的机制仍有待阐明。基因组- Wide RNA定位分析表明，MALAT1与转录活性基因及其前体相互作用。 MRNA.此外，MALAT1与前mRNA剪接因子SR家族的几个成员相互作用 (SRSFs)和MALAT1表达下调的细胞在SRSF介导的AS中表现出缺陷。这个 本研究的目的是阐明MALAT1在SRSF介导的AS中的分子功能。 以低氧反应作为实验模型系统。中心假说是MALAT1通过 在核斑点中浓缩SRSFs，控制SRSFs与其靶前mRNAs和其他 SRSF互动者。在强劲的初步数据的指导下，这一假说将在以下具体情况下进行检验 目的：1)确定MALAT1如何调控SRSF介导的选择性剪接(AS)。2)确定 MALAT1核斑点丰富在前mRNA加工中的意义。在第一个目标中，Pi将 确定MALAT1如何调节SRSF1(SRSF的原型成员)的绑定和招募 蛋白质)与低氧乳腺癌细胞的靶基因前-mRNAs结合。PI还将决定是否参与 MALAT1在小鼠肿瘤模型体内缺氧反应中的作用。在第二个目标PI下，通过使用 超分辨率和实时成像研究将确定MALAT1在空间上的参与 斑点成分的组织，包括SRSFs，以及2)斑点中基因的调控定位 接近。这种方法在技术上是创新的，因为它使用了最先进的技术，包括 超分辨率成像和CRISPR/dCasRx介导的RNA拴系分析。拟议的研究是 重要的是因为破译MALAT1在调节低氧反应的表达中的作用 基因将在乳腺癌治疗的背景下具有广泛的翻译意义。归根结底，这 这些知识将为未来利用MALAT1作为抗癌新的治疗靶点的研究铺平道路。

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 lncRNA that promotes breast tumor metastasis. MALAT1 is induced several folds during hypoxia, and influences the pre-mRNA alternative splicing (AS) of genes controlling hypoxia response. However, the molecular mechanisms by which MALAT1 controls AS during hypoxia signaling remain to be elucidated. Genome- wide RNA mapping analyses reveal that MALAT1 interacts with transcriptionally active genes and their pre- mRNA. Further, MALAT1 interacts with several members of the SR-family of pre-mRNA splicing factors (SRSFs), and cells with deregulated expression of MALAT1 show defects in SRSF-mediated 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, controls the binding of SRSFs with their target pre-mRNAs and other SRSF interactors. 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 pre-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 involvement of MALAT1 in AS during in vivo hypoxia response in 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, including SRSFs, and 2) regulated localization of genes in speckle proximity. The approach is technically innovative, because it employs state of the art techniques, including super-resolution imaging and CRISPR/dCasRx-mediated RNA tethering assays. The proposed research is significant because deciphering the role of MALAT1 in regulating the expression of hypoxia responsive 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.