- non-coding RNAs in the PGCs of male mice, spermatogonia, gametes and in zygotes. - Results: Here, using specific bioinformatics analyses, we have defined the diversity of mitosRNAs present in early differentiated germ cells of male mice (PGCs and spermatogonia), and in the gametes of both sexes and in zygotes.. - We found strong transcription of mitosRNAs relative to the size of the mtDNA, and classifying these mitosRNAs into different functional sncRNA groups highlighted the predominance of Piwi-interacting RNAs (piRNAs) relative to the other types of mitosRNAs. - Their role in oxidative phosphoryl- ation (OXPHOS) has led the mitochondria to be consid- ered as the “powerhouse” of the cell, although they have also been implicated in the regulation of many other pro- cesses, including: apoptosis, calcium homeostasis, aging, signaling, stem cell renewal and immune responses [2, 3].. - In mammals, some mitochondrial genes are contained in specific coding regions of the nuclear DNA but the majority are found in compact circular DNA (1.6 Kb) in- side the mitochondria (mtDNA). - Mitochondrial genes are asymmetrical distributed in the two mtDNA strands and they are transcribed from the PH and PL promoters as a long polycistronic precursor [8]. - (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - In fact, studies of mitochondrial miR- NAs in different species have established the idea that miRNAs and the RISC machinery interact in the mito- chondria, designating these as mito-miRNAs [16, 17].. - PiRNA sequences produced by the mitochondrial genome have since been identified [21], although there is still no evidence of piRNAs or PIWI proteins in the mitochondria of mouse cells to date. - However, PIWIL-1, a human homolog of the mouse MIWI protein, has been detected in the mitochondria of human cancer cells [21].. - NGS analyses of the sncRNAs in mouse male PGCs at E13, spermatogonia cells (SPG), spermatozoa (SPZ), oo- cytes (OCY) and zygotes (ZYGO) suggested important regulatory roles of these sncRNAs in fertilization and gamete differentiation [13, 22]. - Although these mitochondrial sncRNAs reads represent less than 1% of the total sncRNA reads, we found that the mito- chondria in the mouse cells analyzed were enriched in sncRNA molecules (Additional file 1: Figure S2), with the exception of the SPZ (Table 1), particularly when taking into account the amount of mouse mtDNA (Kbs/. - Table 1 Global analysis of the small RNA population associated with mouse mitochondria. - We performed a read length analyses of the mitosRNAs identified (Fig. - We used a bioinformatics pipeline that incorporated information from different sncRNA databases to classify these mitosR- NAs, which classified 80 to 90% of the mitosRNA reads as piRNAs (Fig. - 2b, Table 2), particularly in the OCY and ZYGO (Fig. - Interestingly, 8% of the mitosRNA population in PGC cells were miRNAs, which. - were less predominant in the other cell types and they represented as little as 0.6% in OCY (Fig. - We detected that more than 70% of the mitosRNAs mapped again mouse nu- clear DNA (Fig. - 2c) in gametes and zygotes, reaching a maximum in zygotes where they represented 81% of the mitosRNA reads. - Analyzing the chromosome distribu- tion of the mitosRNA indicated an enrichment on chromosome 2 for all cell types (Fig. - 2d) and it was noteworthy that mitosRNAs from PGCs had a distinct chromosome profile than in the other cell types (Fig. - Moreover, the analysis of specific nuclear mito- chondria sequences (NUMTs) in the mouse nuclear genome indicated that the distribution of the mitosRNA sequences was not biased to the NUMTs. - 1 The sncRNAs sequence coverages in the mitochondrial genome of male PGCs, spermatogonia, spermatozoa, oocytes and zygotes. - Circular representation of the sncRNA coverage in mouse mtDNA. - The radial bars represent the log transformation of the sncRNA read coverage. - Annotation of the mitochondrial genes (dark blue), and the rRNA (light blue) and tRNA (red) was obtained from the Ensembl database. - Coverage of the piR-7,456,245 region is indicated in the piRNA circle by a black arrow in the corresponding piRNA circle. - These results were consistent with the hypothesis proposing mitosRNAs as key elements in the communication between mitochondria and the nu- cleus [17].. - However, the diversity of the miRNAs, does not presented the same increase (Table 2).. - partial correspondence with the 5′ region of the canonical miRNA (paramiR);. - and partial correspondence with the 3′ region of the ca- nonical miRNA sequences (circumiR). - A hierarchical clustering of the mito-miRNA isoforms reflected specific cell expression patterns (Fig. - Interestingly, more than 90% of the mito-miRNA isoforms in SPZ were classified as paramiRs (Fig. - 3b), mito-miRNAs that retain a partial correspond- ence with the 5 ′ region of the canonical miRNAs.. - 2 Characterization of the mitochondrial sncRNA populations in male PGCs, spermatogonia and spermatozoa, and in oocytes and zygotes. - The percentage of reads was calculated from the total reads in the small RNA-Seq library. - d Chromosome distribution of the mitosRNAs derived from the mouse genome.. - Normalization of the read count was carried out using DESeq. - Different isoforms of mito-miRNAs encoded in the mtDNA displayed cell specific expression and diversity, suggesting a possible function that remains as yet unknown.. - The mitochondrial-associated mmu-miR-6390 isoform Despite the distinct expression of the mito-miRNA iso- forms in different cell types, we identified a specific mito-miRNA isoform expressed in all samples, isomiR-6390 (Fig. - Thus, we identified 46 potential targets of the predicted miRNA:- target interactions in two databases (Additional file 2:. - 3 Analysis of the miRNAs derived from mtDNA in male PGCs, spermatogonia, spermatozoa, and in oocytes and zygotes. - a Non-supervised hierarchical clustering of miRNA and miRNA isoform counts associated with the mitochondrial genome in the different samples. - b Distribution of the different isoforms of miRNAs derived from mtDNA. - MicroRNA isoforms: isomiR, miRNA isoforms with the same seed sequence as canonical and trimmed nucleotides in the 3 ′ region. - c Different isomiRs and paramiRs found in miR-6390 in the different cell types: red bar corresponds to the canonical form of the miR-6390 . - Interestingly, PPAR signaling path- ways participate in the regulation of mitochondrial β-oxidation [26].. - Interestingly, mito-piRNAs were the most predominant mitosRNA population in the mitochondria of these cell types (Fig. - Also, all regions of the mouse mitochondrial genome encoded mito-piRNAs, and both mtDNA strands (Figs. - However, strong expression of mito-piRNAs was detected from the mtDNA re- verse strand in the non-coding region of the D-loop (Figs. - 4 Expression of mitochondrially encoded piRNAs and hallmarks of the mitochondrial piRNA populations from male PGCs, spermatogonia and spermatozoa, and oocytes and zygotes. - b Coverage distribution of the piRNAs from tRNAs in different samples, the graphs representing the median coverage of piRNA reads in the different tRNA samples. - c The read length distribution of the piRNA populations derived from the mtDNA. - d Nucleotide frequency at the first and tenth nucleotide of the piRNA reads. - lowest mito-piRNA coverage in the D-loop region with respect to the other cell types (Fig. - 1, black line in the SPZ circle).. - More than 80% of the mito-piRNAs were derived from 5′ mt-tRNA arms in all cell types (Fig. - Figure S4), and they were more predominant in the PGC and ZYGO samples (Fig. - We performed an analysis of the nucleotide frequency at position 1 and 10 of the mitochondria encoded piRNAs (Fig. - Using an in silico ap- proach, we identified an isoform of the MIWI2 protein (PIWIL4 – 201, Ensembl transcript name) that presented a high probability of localizing to the mitochondria (Additional file 2: Table S3). - To address the potential functions of mito-piRNAs, we applied our bioinformatics pipeline to three small-RNA sequence datasets from 3T3-L1 fibroblasts available in the GEO database (Additional file 2: Table S4). - Interestingly, two of the 3T3-L1 libraries analyzed were enriched in 29 nt reads associated with mtDNA (Additional file 1: Figure S5A) and we found that mito-piRNAs were the most highly represented mitosRNAs in all three libraries (Additional file 1:. - There was a variation in the number of mitosRNAs that map to nuclear DNA in the 3 librar- ies, which was 50% on average (Additional file 1:. - 2) because is encoded in the D-loop region, a key regulatory region of mitochondrial genome [8, 9] (Fig. - Table S1) and AntipiRNA-F was also used to detect the mito-piR-7,456,245 in the mitochondria of 3T3-L1 cells (Fig. - To assess the role of mito-piR-mmu-7,456,245 in mitochondria, we attempted to inhibit its regulatory activity using an Anti-piRNA LNA ™ GapmeR probe, analyzing the bioenergetic properties of the 3T3-L1 cell line. - This failure to alter the respiratory cap- acity of the cells was indicative of the presence of a fully functional mitochondrial mass, although it may reflect the inability of this probe to access the mitochondrial matrix or weak RNase H activity in the mitochondria.. - Indeed, mitochondria fulfill a pivotal role in reproduction, both in the development and maintenance of germ cells and gametes, as well as in successful fertilization and early embryonic development [30]. - As such, piRNAs were the most abundant sncRNA population in the mitochondria of the mouse cells analyzed. - To date, miRNAs are the best studied sncRNAs in the mitochondria [17, 26], although we also identified miRNA sequences encoded by mtDNA and these mito-miRNAs represent a large proportion of the miRNAs isoforms de- tected. - In an attempt to gain some insight into this issue, we performed an in silico target analyses of the mito-miR-6390 isomiR expressed in all the cell types analyzed. - This functional analyses of the isomiR-6390 target genes suggested possible roles in the regulation of fatty acid metabolism via the PPAR signal pathway. - The PIWI biogenetic pathway is associated to mito- chondria organelle [32] and it participates in the forma- tion of a key developmental structure in D. - 5 Quantification and analysis of the mito-piR-7,456,245 derived from mitochondria in 3T3-L1 cells. - The heat map colors correspond to log 2 of the normalized read counts. - The arrows indicate the partial localization of the AntipiRNA-F fluorescent GapmeR and the mitochondria. - In addition, our in silico analyses detected the expression of mito-piRNAs encoded in the non-coding region, such as the D-loop. - Finally, the mito-piR-7,456,245 sequence was present in the mitochondria of 3T3-L1 cells and we focused on this mito-piRNA as it is expressed from the D-loop region, an important non-coding regulatory region [8, 9]. - The mito-piR-7,456,245 sequence was found up-regu- lated in the hippocampus of trained mice [33] and it is one of the most strongly expressed piRNAs in the D-loop region in all cell types. - Nevertheless, this is one of the first studies to characterize piRNAs encoded by mtDNA in mammalian cells [21].. - The mitochondrial nuclear genome is the result of the transfer of mtDNA fragments during evolution, including those in the D-loop region [34, 35]. - The presence of relevant mito-piRNAs is consistent with the germline nature of the cells analyzed and the high proportion of the mitosRNAs associated with the NUMTS identified. - However, the existence and activ- ity of TEs in the mtDNA is more controversial. - Activation of TEs in mtDNA does not appear to be com- mon in animals and consequently, the stability of the mtDNA with respect to transposition activity is high.. - All the mapping steps in the bioinformatics pipeline were performed using the Bowtie aligner (http://bowtie-bio.sourceforge.. - In silico identification and classification of sncRNAs The classification of sncRNAs associated with mitochon- dria in the different databases was performed by sequential mapping using Bowtie, as described in Additional file 1:. - To determine the coverage of the 5′ and 3’ tRNA arm, the average read depth for each fragment was calculated based on the 5′ and 3′ region de- fined by the anticodon in each mitochondrial tRNA.. - Circular plots of the mitochondrial genome associated sncRNAs were created using coverage data from BEDTools and Circleator v1.o (http://jonathancrab tree.github.io/Circleator. - The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a proxy for lactate production) of the cells were recorded to assess the mitochondrial respiratory and glycolytic activity, respectively. - Density of the sncRNA reads per Kb.. - Nuclear mitochondrial sequences (NUMTs) identified in the mouse genome.. - Average nucleotide coverage of the mito-piRNAs in mt-tRNAs.. - In silico analysis of the mitochondrial localization of mouse PIWI protein isoforms. - Description of the small RNA-Seq datasets in 3T3-L1 cells obtained from the GEO database. - Non-coding RNAs: multi-tasking molecules in the cell. - PIWI proteins and PIWI-interacting RNAs in the soma. - Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells. - The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race
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