Chapter 1 is an introduction to the work detailed in this thesis and details the background of the main biological molecules that are studied. The recent discovery of novel long-non coding RNAs (lncRNAs) reveals a new class of molecules with immense biological relevance and promising candidates for therapeutic targeting. Classified as RNA molecules longer than 200 nucleotides in length, lncRNAs have been found to be dysregulated in various disease conditions, and their return to steady-state levels through transcript level manipulation has been observed to induce cell and tissue survival. Of great interest is the lncRNA MALAT1 which is known to be dysregulated in various cancer tissues and neurodegenerative diseases. Localized within the nucleus, MALAT1 is involved in a variety of important biological processes including mRNA transcriptional regulation, alternative splicing, and stabilizing nascent nuclear speckles. A detailed analysis of direct protein binders to MALAT1 reveals TDP-43 as a strong binder with common functionalities present. Both MALAT1 and TDP-43 are dysregulated in neurodegenerative diseases, and their common functionalities overlap in alternative splicing and transcript regulation. An increase in MALAT1/TDP-43 binding is also observed in neurodegenerative diseased tissues, but the reason for this interaction is yet to be explored fully in literature. Thus, it is important to study the common functions of MALAT1 and TDP-43 in the context of neurodegeneration and understand if the MALAT1/TDP-43 complex plays a role in regulating biological mechanisms for cell survivability, which can then be used for therapeutic advancements in this field.

Chapter 2 is focused on the regulation of TDP-43 binding to mRNA targets by MALAT1. The increase in MALAT1/TDP-43 interaction in neurodegenerative disease tissues indicates a potential function for this interaction. Given TDP-43’s known function of regulating gene expression level, we hypothesized that MALAT1 may play a role in regulating TDP-43 function and gene regulation. We found that altering TDP-43 expression alters MALAT1 expression, and disruption of the steady-state level of either transcript induces cell death in neuroblastoma samples. TDP-43 binds to the 3’UTR of mRNA genes, and reduced MALAT1 expression can increase the binding ability of TDP-43 to target 3’UTR genes in HEK293 and SH-SY5Y cells. This suggests that MALAT1 regulates the binding activity of TDP-43 to target genes in the nucleus. This hypothesis is extended in a Parkinson’s Disease model through the induction of MPP+ to SH-SY5Y cells, and a similar alteration in TDP-43 binding and gene expression regulation is observed in altered MALAT1 conditions. From the collected data, we can infer the existence of a coordinated network of RNA-protein binding that mediates gene expression levels and ensures homeostatic concentrations in cell. Dysregulation of the MALAT1 lncRNA results in aberrant TDP-43 function, which has severe consequences for cell viability.

Chapter 3 details the role of MALAT1 in regulating alternative splicing through sequence-specific binding interactions. Alternative splicing (AS) regulation is a common function of both TDP-43 and MALAT1, and the increase in TDP-43/MALAT1interaction may affect gene expression level and molecular pathway regulation through changes in AS. We hypothesized that MALAT1 and TDP-43 may concurrently regulate gene expression of a common splice target, the spermine/spermidine acetyltransferase 1 (SAT1) pre-mRNA. Further investigation revealed the existence of overlapping sequence-specific binding regions between MALAT1/SAT1 and MALAT1/TDP-43 that regulated SAT1 AS. These regions help efficiently recruit SAT1 pre-mRNA to TDP-43, with MALAT1 acting as a bridge to direct localization. This is a novel proposed mechanism for AS regulation by lncRNA. We then extended this hypothesis to another splicing factor and pre-mRNA target and observed a similar recruitment phenomenon for the MALAT1/CSTF2/PPFIA3 interactome. We hence present a model in which MALAT1 can recruit both pre-mRNA and splicing factor protein via an overlapping binding region with distinct sequence-specific targeting for efficient AS.