Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’.
Highlights
Mitochondria are eukaryotic organelles that maintain and express their own genome, known as the mitochondrial DNA
The full set of mitochondrial transfer RNAs and two ribosomal RNAs, namely the 12S and 16S rRNAs, are transcribed from the mitochondrial DNA (mtDNA) [2], while the RNA moiety of the RNase MRP enzymes [3,4,5], the 5S rRNA [6,7], and two species of tRNAGln [8] are all RNAs delivered into mitochondria from the nucleus
Mitochondrial fraction was analyzed for its purity by immunoblot as assessed by the mitochondrial marker ATP synthase subunit a 1 (ATP5A1) and the nuclear/cytosolic marker cyclin-dependent kinase 2 (CDK2), which indicated the lack of nuclear and cytosolic contaminants reproducibly (Figure 1A)
Summary
Mitochondria are eukaryotic organelles that maintain and express their own genome, known as the mitochondrial DNA (mtDNA). The transcription and translation of the mtDNA as well as the processing of mitochondrial transcripts requires the involvement of several types of non-coding RNAs (ncRNA), which can be either mitochondrially encoded or transcribed within the nucleus and subsequently localized to mitochondria [1]. MiRNAs are transcribed within the nucleus and are extensively processed and matured in the cytosol as ,22-bp double-stranded RNA. RNAi occurs upon pairing one of the two miRNA strands, embedded in an AGO protein, with target sites in an mRNA, thereby affecting the stability/translation of this mRNA [11]. Knockdown and knockout of AGO2, respectively in human cells and in mice, suggest that the protein may have specific functions that may not be complemented by the other AGOs [13,15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
