Nuclear miRNA regulates the mitochondrial genome in the heart

Abstract

Rationale: Mitochondria are semiautonomous cellular organelles with their own genome, which not only supply energy but also participate in cell death pathways. MicroRNAs (miRNAs) are usually 19 to 25 nt long, noncoding RNAs, involved in posttranscriptional gene regulation by binding to the 3'-untranslated regions of target mRNA, which impact on diverse cellular processes.

Objective: To determine if nuclear miRNAs translocate into the mitochondria and regulate mitochondrial function with possible pathophysiological implications in cardiac myocytes.

Methods and results: We find that miR-181c is encoded in the nucleus, assembled in the cytoplasm, and finally translocated into the mitochondria of cardiac myocytes. Immunoprecipitation of Argonaute 2 from the mitochondrial fraction indicates binding of cytochrome c oxidase subunit 1 (mt-COX1) mRNA from the mitochondrial genome with miR-181c. Also, a luciferase reporter construct shows that mi-181c binds to the 3'UTR of mt-COX1. To study whether miR-181c regulates mt-COX1, we overexpressed precursor miR-181c (or a scrambled sequence) in primary cultures of neonatal rat ventricular myocytes. Overexpression of miR-181c did not change mt-COX1 mRNA but significantly decreased mt-COX1 protein, suggesting that miR-181c is primarily a translational regulator of mt-COX1. In addition to altering mt-COX1, overexpression of miR-181c results in increased mt-COX2 mRNA and protein content, with an increase in both mitochondrial respiration and reactive oxygen species generation in neonatal rat ventricular myocytes. Thus, our data show for the first time that miR-181c can enter and target the mitochondrial genome, ultimately causing electron transport chain complex IV remodeling and mitochondrial dysfunction.

Conclusions: Nuclear miR-181c translocates into the mitochondria and regulates mitochondrial genome expression. This unique observation may open a new dimension to our understanding of mitochondrial dynamics and the role of miRNA in mitochondrial dysfunction.

Figure 1. RNA isolation and identification of miRNA in heart-derived mitochondria

(A) Capillary electrophoresis of total heart vs. mitochondrial fraction. (B) Gel electrophoresis of mitochondrial RNA (marked in RED) vs. total heart. (C) Heat Map representation of the miRNA microarray analysis in Mitochondrial Fraction (Mt) and Total Heart Homogenate (Total), using Affymetrix Chip. The log2 expressions of each gene are mean-centered, based on all the data from that gene.

Figure 2. Mitochondrial localization of miR-181c

qRT-PCR shows that miR-181c is mainly present in the mitochondria. (A) miR-181c expression is almost the same in total RNA derived from the mitochondrial fraction and the total heart fraction. 12S rRNA is the internal control to normalize these data. (B) miR-181c expression is mainly detected in the mitochondrial fraction and not in the cytosolic fraction (left panel), whereas miR-1191 is mainly present in the cytosolic fraction (right panel). 5S rRNA is the internal control to normalize these data, as this RNA is present in both cytosol and mitochondrial fractions. *p<0.05 vs. Cytosol. (C) Fluorescent in situ hybridization demonstrating intramitochondrial localization of miR-181c. Mitochondria are labeled with mito-tracker red (left), miR-181c is green (GFP, middle), and the merged image is shown in the right panel.

Figure 3. Active involvement of miR-181c in the mitochondrial RISC complex

(A) Western blot shows that only Ago 2, and not Dicer or TRBP, is present in the mitochondria. Prohibitin is used as a mitochondrial marker. qRT-PCR shows both (B) miR-181c and (C) mt-COX1 mRNA, and no other mitochondrial gene products, like (D) mt-COX2 mRNA or (E) 12S rRNA; in the Ago 2 immunoprecipitate from the mitochondrial fraction.

Figure 4. Biogenesis and target of miR-181c

(A) qRT-PCR shows the amounts of pre-miRNA-181c in the total heart sample vs. the different fractions. *p<0.05 vs. Total Heart and † p<0.05 vs. Nucleus. (B) Luciferase activity derived from the COX-1 3′ UTR reporter following transfection into HeLa cells with negative or miR-181c mimic. All values were normalized to Renilla luciferase activity produced from a cotransfected control plasmid. *p<0.05 vs. Neg Control. (C) Neonatal cardiomyocytes were transfected with precursor miR-181c or a scrambled sequence. qRT-PCR shows increased miR-181c after 48 hours. 12S rRNA served as the internal control to normalize these data. *p<0.05 vs. Neg Control.

Figure 5. Target of miR-181c in the mitochondrial genome

(A) Overexpression of miR-181c does not change the steady state level (left panel) of mt-COX1 mRNA but mt-COX1 protein is significantly reduced (right panel). Western blot of mt-COX1 and gel densitometry analysis is shown. Content of mt-COX1 was normalized to Cadherin and 12S rRNA was used as internal control for qRT-PCR. *<p 0.05 vs Neg Control. Mean ± SEM (n=4) are shown. (B) Overexpression of miR-181c increased mt-COX2 mRNA (left panel) and protein expression (right panel) compared to negative control. Content of mt-COX2 protein was normalized to Cadherin and 12S rRNA was used as internal control for qRT-PCR. *

Figure 6. Effect of miR-181c on mitochondrial function

(A) Myocytes were transfected to overexpress miR-181c, or negative control. Cells were permeabilized and oxygen consumption measured after adding substrate for complex IV, TMPD/Ascorbate. (*p<0.05 vs. Neg Control, n=6) (B) Time course of ROS generation from myocytes overexpressing miR-181c. (*p<0.05 vs. Neg Control., n=4). (C) Over-expression of miR-181c significantly increases the rate of ROS production compared to the negative (Neg) control group. *p<0.05 vs. Neg Control n=4.