The landscape of mitochondrial small non-coding RNAs in the PGCs of male mice, spermatogonia, gametes and in zygotes

Abstract

Background: Mitochondria are organelles that fulfill a fundamental role in cell bioenergetics, as well as in other processes like cell signaling and death. Small non-coding RNAs (sncRNA) are now being considered as pivotal post-transcriptional regulators, widening the landscape of their diversity and functions. In mammalian cells, small RNAs encoded by the mitochondrial genome, mitosRNAs were discovered recently, although their biological role remains uncertain.

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. Mito-piRNAs were more abundant in oocytes and zygotes, where mitochondria fulfill key roles in fecundation process. Functional analysis of some particular mito-piRNAs (mito-piR-7,456,245), also expressed in 3T3-L1 cells, was assessed after exposure to RNA antagonists.

Conclusions: As far as we are aware, this is the first integrated analysis of sncRNAs encoded by mtDNA in germ cells and zygotes. The data obtained suggesting that mitosRNAs fulfill key roles in gamete differentiation and fertilization.

Keywords: Gametes; Mitochondria; Primordial germ cells; Small non-coding RNA; Zygotes.

Fig. 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. Reads from: primordial germ cells (PGC) are represented in red; spermatogonia (SPG) in blue; spermatozoa (SPZ) in green; oocytes (OCY) in purple; and zygotes (ZYGO) in gold. Annotation of the mitochondrial genes (dark blue), and the rRNA (light blue) and tRNA (red) was obtained from the Ensembl database. The read coverage was obtained using BedTools software and the circular representation was created using the Circleator tool. Coverage of the piR-7,456,245 region is indicated in the piRNA circle by a black arrow in the corresponding piRNA circle

Fig. 2

Characterization of the mitochondrial sncRNA populations in male PGCs, spermatogonia and spermatozoa, and in oocytes and zygotes. a Read length distribution of mitochondrial derived sncRNAs from different cell types. The percentage of reads was calculated from the total reads in the small RNA-Seq library. b Classification of mitosRNAs in microRNAs (miRNA - mito-miRNAs), PIWI-interacting RNAs (piRNAs - mito-piRNAs) and sequences from non-coding RNAs present in the Ensembl database (ncRNAs). Reads that do not map to previous databases are considered as not annotated. c The percentage of mitochondrial encoded sncRNAs that map to the mouse genome (MM10 - including nuclear mitochondrial sequences) and those exclusive to mitochondrial DNA (MT). d Chromosome distribution of the mitosRNAs derived from the mouse genome. Normalization of the read count was carried out using DESeq. Male cells: primordial germ cells (PGCs), spermatogonia cells (SPG), and spermatozoa (SPZ). Female cells: oocytes (OCY) and zygotes (ZYGO)

Fig. 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. Read counts were normalized using DESeq and transformed to a base two logarithm. The annotation corresponds to the pre-microRNA and the miRNA isoforms. 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; paramiR, miRNA isoform where the 5′ region corresponds to the canonical miRNA sequences; and circumiR, the miRNA isoform where the 3′ region corresponds to the canonical miRNA sequences. 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; the asterisks indicate the most expressed miRNA isoform in each sample. d Enrichment of KEEG pathways among the miR-6390 targets. Male cells: primordial germ cells (PGCs), spermatogonia cells (SPG), and spermatozoa (SPZ). Female cells: oocytes (OCY) and zygotes (ZYGO)

Fig. 4

Expression of mitochondrially encoded piRNAs and hallmarks of the mitochondrial piRNA populations from male PGCs, spermatogonia and spermatozoa, and oocytes and zygotes. a Expression heatmap of piRNAs generated from different mitochondrial genes where the heatmap color indicates the normalized expression of piRNAs derived from mitochondrial genes. The annotation of mitochondrial genes was obtained from Ensembl database. 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. Schematic representation of tRNA arms. c The read length distribution of the piRNA populations derived from the mtDNA. The read percentage was calculated using the total number of piRNA reads in each sample. d Nucleotide frequency at the first and tenth nucleotide of the piRNA reads. Male cells: primordial germ cells (PGCs), spermatogonia cells (SPG), and spermatozoa (SPZ). Female cells: oocytes (OCY) and zygotes (ZYGO)

Fig. 5

Quantification and analysis of the mito-piR-7,456,245 derived from mitochondria in 3T3-L1 cells. a Expression of 7 selected piRNAs in NGS libraries. The heat map colors correspond to log₂ of the normalized read counts. b The RT-qPCR expression of 7 selected piRNAs in arbitrary units calculated using U6 derived piRNA as an endogenous standard. c Confocal images of cultured 3T3-L1 cells transfected with AntipiRNA-F fluorescent GapmeR, and stained with MitoTracker (mitochondria) and DAPI (nucleus). d Merged image of 3T3-L1 cells transfected with AntipiRNA-F fluorescent GapmeR, and stained with MitoTracker and DAPI. The arrows indicate the partial localization of the AntipiRNA-F fluorescent GapmeR and the mitochondria. Male cells: primordial germ cells (PGCs), spermatogonia cells (SPG), and spermatozoa (SPZ); female cells: oocytes (OCY); and zygotes (ZYGO). Bars represent 75 μm