Guidelines for mitochondrial RNA analysis

Abstract

Mitochondria are the energy-producing organelles of mammalian cells with critical involvement in metabolism and signaling. Studying their regulation in pathological conditions may lead to the discovery of novel drugs to treat, for instance, cardiovascular or neurological diseases, which affect high-energy-consuming cells such as cardiomyocytes, hepatocytes, or neurons. Mitochondria possess both protein-coding and noncoding RNAs, such as microRNAs, long noncoding RNAs, circular RNAs, and piwi-interacting RNAs, encoded by the mitochondria or the nuclear genome. Mitochondrial RNAs are involved in anterograde-retrograde communication between the nucleus and mitochondria and play an important role in physiological and pathological conditions. Despite accumulating evidence on the presence and biogenesis of mitochondrial RNAs, their study continues to pose significant challenges. Currently, there are no standardized protocols and guidelines to conduct deep functional characterization and expression profiling of mitochondrial RNAs. To overcome major obstacles in this emerging field, the EU-CardioRNA and AtheroNET COST Action networks summarize currently available techniques and emphasize critical points that may constitute sources of variability and explain discrepancies between published results. Standardized methods and adherence to guidelines to quantify and study mitochondrial RNAs in normal and disease states will improve research outputs, their reproducibility, and translation potential to clinical application.

Keywords: MT: Non-coding RNAs; gene expression; guidelines; microRNAs; mitochondria; noncoding RNAs.

Graphical abstract

Figure 1

Biogenesis and targeting of mitochondria-related ncRNAs Mitochondria-related ncRNAs are of nuclear (nDNA) or mitochondrial DNA (mtDNA) origin. Nuclear genome-encoded lncRNAs and miRNAs are being processed in the cytosol and regulate mitochondrial physiology (anterograde signaling) by targeting transcripts/proteins outside or inside the organelle. The mechanism of import of ncRNAs in the mitochondrial matrix remains elusive. piRNAs encoded by the nuclear genome are being processed by MitoPLD at the surface of mitochondria. The mitochondrial genome encodes lncRNAs, miRNAs, piRNAs, and circRNAs that regulate mitochondrial and nuclear (retrograde signaling) gene expression. Whether mitochondria are autonomous for the maturation of these ncRNAs or they depend on cytosolic enzymes is a matter of debate (diagrams were adapted with permission from Servier Medical Art library, available under Creative Commons license).

Figure 2

Workflow of mitochondrial RNA expression profiling (A) Isolation of mitochondria by differential centrifugation, ultra-centrifugation using density gradients, or antibody-mediated capture using magnetic separation (MACS). (B) Mitochondria purification after RNase A treatment to remove cytosolic RNAs from the outer mitochondrial membrane. (C) Quality control of mitochondrial preparations using enzymatic assays, microscopy, and enrichment analysis of mitochondrial markers. (D) Mitochondrial RNA extraction and quality control. (E) Gene expression analyses including RNA sequencing and RT-qPCR. (F) Assessment of mitochondrial function (created with BioRender.com).

Figure 3

In silico analysis of mitochondria-related ncRNAs Possible approaches to retrieve expression profiles of mitochondria-related RNAs from bulk transcriptomic data. Appropriate functional tests will confirm the biological relevance of the differentially expressed miRNAs and validate the link between these miRNAs and their predicted mRNA targets.