UC San Diego

Non-coding RNA transcripts regulate diverse functions including gene expression, splicing, and cell growth in human cells through their interactions with RNA binding proteins. The full biological relevance of these RNA/protein interactions and the role of RNA in regulating protein function is still unclear in many cases. This work investigates two specific cases of lncRNA regulating gene expression to better understand the cellular functions and molecular details of lncRNA/protein interactions. Specifically, the ability of the lncRNAs, MALAT1 and XIST, to regulate gene expression through their interactions with the RNA binding proteins TDP-43 and SHARP, respectively, is examined in this thesis. Chapter 2 is focused on the interaction of TDP-43 protein and MALAT1 non-coding RNA. TDP-43 protein is associated with neurodegenerative disease and has been shown to bind directly to MALAT1 non-coding RNA. We investigated the dependence of TDP-43 localization to the 3’ untranslated regions (3’ UTRs) of other mRNAs upon the level of MALAT1 expression in human cells. Target RNAs selected for analysis were representative of high, moderate, and low expression mRNAs bound by TDP-43 in multiple previous CLIP-seq studies. To measure the affinity of TDP-43 binding to MALAT1 long noncoding RNA and 3’ UTR mRNA targets, we used a newly designed fluorescent multiplexed version of the electrophoretic mobility shift assay (mEMSA), described in Chapter 3. Binding affinities of TDP-43 for mRNA 3’ UTRs correlated positively with the number of UG repeats in the identified CLIP-seq peak binding region. In addition, CLIP-seq peak signal height directly correlated with TDP-43 in vitro binding affinity for mRNA 3’ UTR targets. The binding of TDP-43 protein to messenger RNA 3’ UTR targets was evaluated by immunoprecipitation and quantitative real-time PCR (RT-qPCR). Decreased MALAT1 expression resulted in re-localization of TDP-43 protein to mRNA 3’ UTRs. TDP-43 binding stabilized the mRNA expression levels of the mRNA transcripts. Neuroblastoma cells showed an upregulation of MALAT1, downregulation of mRNA targets, and increased cell death in response to MPP+ treatment, a cellular condition modeling Parkinson’s disease. Knockdown of MALAT1 reversed these results, indicating that the MALAT1/TDP-43 interaction contributes directly to cell death. Our data suggest that MALAT1 lncRNA expression is required to maintain the correct balance of TDP-43 binding to messenger RNA 3’ UTR targets in mammalian cells. These studies confirm the interdependence of TDP-43 and MALAT1 functions and suggest that mechanisms of neurodegenerative disease may depend in part on the dysregulation of non-coding RNA transcripts. Chapter 3 describes an updated, multiplexed method of the electrophoretic mobility gel-shift assay which can be used for measuring the binding affinities (Kds) for several RNAs in a single gel. This method is performed by first fluorescently labeling each RNA of interest with a unique fluorophore. RNA and protein binding partners are mixed, incubated, and separated by acrylamide gel electrophoresis. The binding of RNAs labeled with different fluorophores can then be measured simultaneously using a fluorescent imaging system, allowing the calculation of a dissociation constant (Kd) for each pair of RNA-protein interactions. This chapter outlines the mEMAS method and validates the approach with specific results for three RNA binding partners of TDP-43 (positive control, TARDBP, TDP-43’s own 3’UTR which has previously been shown to bind TDP-43 in vitro, the negative control BARD1, a region from a non-interacting mRNA, and CSNK1E, a known TDP-43 interactor without a previously published Kd). We found that this multiplexed method allows more rapid analysis and direct comparison of Kd measurements for multiple RNAs with the same protein. Results of the mEMSA approach are also found in Chapters 2 and 4. Chapter 4 provides another specific example of lncRNA/RBP interactions regulating gene expression. XIST non-coding RNA promotes the initiation of X chromosome silencing by recruiting the protein SHARP (SMRT and HDAC Associated Repressor Protein) to one X chromosome in female mammals. SHARP recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the silenced X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We show that binding of SHARP to XIST RNA requires RRM4 of the protein, in contrast to the requirement of RRM3 for specific binding to SRA RNA. Evaluation of SHARP binding to full-length, dimeric, or truncated versions of the A-repeat region revealed that high affinity binding of XIST to SHARP requires only four A-repeat segments. SHARP binding to XIST A-repeat RNA results in changes in the flexibility and structure of the RNA, reflected in chemical structure probing data, and an apparent conformational change of the XIST RNA when bound to SHARP protein by electron microscopy. These findings provide new information on the molecular basis of the XIST and SHARP interaction and how specificity in RNA-protein binding is achieved, furthering our understanding of the mechanisms ensuring proper gene dosage in mammals.