Molecular and cellular role of RNA-binding proteins in cardiac biology and disease
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2022
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Louis, Lithin
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Conventional RNA-binding proteins (RBPs) regulate post-transcriptional gene expression, forming dynamic ribonucleoprotein complexes via their globular RNA-binding domains (RBDs). However, unbiased identification using RNA interactome capture detected multiple RBPs devoid of classical RBDs, exhibiting novel modes of RNA interaction. Cardiomyocyte interactome capture using the murine HL-1 cardiomyocyte cell line detected multiple such unconventional RBPs, notably a high representation of metabolic enzymes. Interacting RNAs could potentially regulate their enzymatic activity or could act as scaffolds facilitating the formation of metabolons. Similarly, the calcium channel protein Sarco-(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), which plays a critical role in regulating calcium homeostasis, was also identified to interact with RNA through an unconventional RBD.
To explore the relevance of such unconventional interactions, I first established an RNA interactome capture protocol using native cardiomyocytes (NCMs) isolated from adult murine hearts to verify the RNA interaction of the metabolic enzymes in a native cardiac context. Based on the interactome capture experiment, the following enzymes that exhibited RNA interaction were selected. Aldolase A (ALDOA), Enolase 1 (ENO1), Phosphoglycerate Kinase 1 (PGK1) and Glucose phosphate isomerase 1 (GPI1) from glycolysis, and Malate dehydrogenase 2 (MDH2), Isocitrate dehydrogenase 2 (IDH2) and Aconitase 2 (ACO2) from citric acid cycle. I also included SERCA2a in this thesis for its critical role in cardiovascular disease and therapeutics.
To identify their interacting RNA targets, I set up an unbiased methodology employing the principles of crosslinking and immunoprecipitation (CLIP) followed by sequencing (CLIP-seq). The CLIP-seq methodology was successfully tested using the conventional RBP PUM2 as a positive control and identified 60% of the known PUM2 RNA targets with a fold change of more than 2 and with a P-value below 0.05. However, at the level of sequencing depth achieved, potential RNA targets of the selected enzymes and SERCA2a, though they displayed a fold change above 2, failed to exhibit P-values below 0.05 and hence will require further experimental validation. Under this proviso, ALDOA, ENO1, PGK1 and GPI1 displayed a positive enrichment for multiple RNAs (16, 20, 11 and 15 RNA targets respectively). EGFP tagged ALDOA and PGK1 shared two common RNA targets, ANKRD40 and RHOQP1.Similarly, CLIP-seq of native SERCA2a from HL-1 cardiomyocytes identified 15 RNA targets. Validation of selected RNA targets (5 out of 15) based on cardiac and associated disease relevance using CLIP-qPCR exhibited positive enrichment for CALD1 when compared to IgG control.
This thesis sought to explore the relevance of selected unconventional RBPs by identifying their potential interacting RNA targets. Interactome capture validated RNA binding by several metabolic enzymes in a native cardiac context and a CLIP-seq methodology was developed to identify their interacting RNA targets. Preliminary results indicated that metabolic enzymes interact with multiple candidate RNAs, exhibiting a common functional theme. Interestingly, the common RNA targets identified for PGK1 and ALDOA suggest the possibility of RNAs facilitating the formation of metabolons enhancing metabolic activity. Whether the RNAs regulate the enzymatic activity or the metabolic enzymes regulate a regulon of mRNAs with similar function, (such as cell proliferation associated mRNAs) is yet to be determined. RNA targets of SERCA2a indicate their involvement in disease and their probable role in regulating the transporter function of the protein. The potential RNA targets identified provide a starting point to explore the sequence motifs and protein domains that determine the interaction. Modulation of the motifs and domains will invariably enable us to determine the biological relevance of the interactions.
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