Characterization of nuclear-retained RNA-mediated gene regulatory mechanisms

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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.