Characterization of nuclear-retained RNA-mediated gene regulatory mechanisms

点击翻译按钮获取中文摘要

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.