MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis

Abstract

MicroRNAs (miRNAs) are a class of small approximately 22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan 5'nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they might serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.

Figure 1

Western blot analysis of Percoll gradient isolated mitochondria. (A) Total proteins (40 μg) isolated from the various fractions during the mitochondria purification process were separated by SDS-PAGE. Following electrophoretic transfer onto nitrocellulose membrane, the immunoblots were processed for western blot analysis to assess purity using primary antibodies against lactate dehydrogenase (LDH, bottom) as a cytosolic marker; cytoplasmic large ribosomal protein L26 for polysomal contamination (middle); and protein CoxIV as a mitochondrial marker (top). Each of the proteins was detected by ECL. The protein is indicated at left, and the molecular weight (MW) in kDa at right. Lane 1, isolated rat liver polysomes; lane 2, whole liver homogenate; lane 3, supernatant post 600 ×g centrifugation; lane 4, supernatant post 1,100 ×g centrifugation; lane 5, pellet from 1,100 ×g centrifugation; lane 6, supernatant post 7,600 ×g centrifugation; lane 7, crude mitochondrial pellet from 7,600 ×g centrifugation; lane 8, mitochondrial fraction from Percoll® gradient; and lane 9, mitochondrial fraction from Percoll® gradient following RNase treatment. (B) Total proteins (25 μg) isolated from the various fractions during the mitochondria purification were separated by SDS-PAGE and processed for immunoblot analysis using primary antibodies anti-LAMP1 (upper) and anti-calnexin (lower) as markers for lysosomal and endoplasmic reticulum, respectively. The proteins were detected using ECL and their identity is shown at left and their molecular weight (MW) in kDa at right. Lane 1, MCF-7 whole cell lysate; lane 2, whole liver homogenate; lane 3, supernatant post 600 ×g centrifugation; lane 4, supernatant post 1,100 ×g centrifugation; lane 5, pellet from 1,100 ×g centrifugation; lane 6, supernatant post 7,600 ×g centrifugation; lane 7, crude mitochondrial pellet from 7,600 ×g centrifugation; lane 8, mitochondrial fraction from Percoll® gradient; and lane 9, mitochondrial fraction from Percoll® gradient following RNase treatment.

Figure 2

Transmission electron micrographs of purified liver mitochndria. Following the Percoll® gradient isolation and washing, a portion of the mitochondrial pellet was fixed overnight at 4°C in cacodylate buffered 6% glutaraldehyde, then processed and embedded for electron microscopy. Sections 70–100 nm in thickness were cut and stained with uranyl acetate and lead citrate. The morphology and purity of the isolated mitochondria were examined using a JEOL-100 CX electron microscope. The representative digital micrographs indicated that the mitochondria fields did not contain significant amounts of non-mitochondrial material (a), and exhibited classical mitochondrial morphology (b and c). (d) The same Percoll® gradient isolated mitochondria post-treatment with RNase showing minimal loss of mitochondrial structural integrity. The length in μm of the scale bars shown is indicated above the bar.

Figure 3

Functional analyses of mitochondrial-associated miRNA. MiRanda and TargetScan algorithm miRNA target predictions for each miRNA were submitted for functional enrichment analyses to Ingenuity Pathways Analyses individually and as a group to identify over-representation of functional groups in the predicted gene set. The heat map shows annotations with p-values less then 10E-6 in 3 or more separate analyses. The miRNA species are indicated at top, representing the results of miRNA set analyses from each predictive algorithm. The results of the analysis of intersection between the two mitochondrial sets is labeled (top) and the functional groups listed immediately left of the map. The color scale for p-values is shown at left.