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A genome-scale analysis of mitochondrial-associated mRNAs in plants: upstream AUGs in 5’UTRs alter mRNA targeting

ABSTRACT: Intracellular sorting of mRNAs is an essential process to regulate gene expression and protein localization. Most of mitochondrial proteins are nuclear-encoded and imported into mitochondria, through post-translational or co-translational processes. To determine cytosolic mRNAs targeted to the mitochondrial surface, a genome-scale analysis of mRNAs associated with mitochondria has been performed in plants. As expected, many messengers encoding mitochondrial proteins were found associated with mitochondria, but numerous mRNAs encoding cytosolic proteins were also detected. This suggests multiple roles for mRNA targeting, in addition to the co-translational import of mitochondrial proteins. Moreover, correlations between mRNA targeting and function of mitochondrial proteins indicate a coordinated control of mRNAs localization within metabolic pathways. At last, upstream AUGs in 5'UTR, known to regulate translation efficiency of downstream sequences, affect mRNA targeting. A mutational approach coupled with in vivo mRNA visualization confirms this observation, and highlights the role of upstream AUGs as regulators of mRNA targeting to the mitochondrial surface.

ORGANISM(S): Solanum tuberosum

PROVIDER: GSE91388 | GEO | 2018/08/01

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein 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. 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’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.

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A genome-scale analysis of mitochondrial-associated mRNAs in plants: upstream AUGs in 5’UTRs alter mRNA targeting

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