Mitochondrial Import of Malat1 Regulates Cardiac Mitochondrial Function in Type 2 Diabetes Mellitus through Interaction with MicroRNA-23b

Abstract

Background: The pathogenesis of type 2 diabetes mellitus is known to alter cardiac mitochondrial bioenergetics, but the role of non-coding RNAs (ncRNAs) in regulating this process remains poorly understood, specifically within the mitochondrion. Here we characterized the mitochondrial localization of nuclear-encoded lncRNAs in the heart in an effort to understand how import of lncRNAs can modify mitochondrial function during diabetes mellitus.Methods: Human patient and FVB/NJ mouse cardiac tissues were collected from type 2 diabetics and non-diabetics. Immunoblotting was used to validate purity of mitochondrial fractionation. NcRNA from subcellular compartments (cytoplasmic and mitochondrial) were sequenced through the Illumina 2500 HiSeq. LncRNA mitochondrial targeting sequences were acquired through crosslinking immunoprecipitation (CLIP) with Polynucleotide Phosphorylase (PNPase) and secondary structures were analyzed using supervised and unsupervised machine learning algorithms. Fluorescence in situ hybridization (FISH) allowed for visualization of Malat1 localization. Mitochondrial function was assessed through oxygen consumption rate.Results: 2448 LncRNAs were discovered in human cardiac mitochondria. Malat1 and microRNA-23b abundance were significantly reduced in human and mouse diabetic mitochondria. Modification of the import protein PNPase in HL-1 cells decreased binding affinity (~80%) of transcript variant 4 of Malat1 (Malat1-204), with machine learning (AUC 0.75) identifying stem-loops as shared secondary structures of lncRNAs bound to PNPase. Knockdown of Malat1-204 and microRNA-23b in mitochondria decreased protein expression of mt-Nd4 (~55%), though showed little/no binding homology to the mt-Nd4 mRNA transcript. Malat1-204 and microRNA-23b were identified bound to mt-Rnr1 rRNA, which could influence translation of mt-Nd4 through the mitochondrial 12S rRNA pathway.Conclusions: Malat1-204 and microRNA-23b, decreased in cardiac diabetic mitochondria, likely stabilize the mitochondrial ribosomal complexes through binding of mt-Rnr1. The significant decrease in total abundance of lncRNAs in diabetic mitochondria could suggest maladaptation for regulating bioenergetics and managing future insults.