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. 2019 May;14(5):504-521.
doi: 10.1080/15592294.2019.1600389. Epub 2019 Apr 8.

Somatic Expression of piRNA and Associated Machinery in the Mouse Identifies Short, Tissue-Specific piRNA

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Free PMC article

Somatic Expression of piRNA and Associated Machinery in the Mouse Identifies Short, Tissue-Specific piRNA

Bambarendage P U Perera et al. Epigenetics. .
Free PMC article

Abstract

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that associate with PIWI proteins for transposon silencing via DNA methylation and are highly expressed and extensively studied in the germline. Mature germline piRNAs typically consist of 24-32 nucleotides, with a strong preference for a 5' uridine signature, an adenosine signature at position 10, and a 2'-O-methylation signature at the 3' end. piRNA presence in somatic tissues, however, is not well characterized and requires further systematic evaluation. In the current study, we identified piRNAs and associated machinery from mouse somatic tissues representing the three germ layers. piRNA specificity was improved by combining small RNA size selection, sodium periodate treatment enrichment for piRNA over other small RNA, and small RNA next-generation sequencing. We identify PIWIL1, PIWIL2, and PIWIL4 expression in brain, liver, kidney, and heart. Of note, somatic piRNAs are shorter in length and tissue-specific, with increased occurrence of unique piRNAs in hippocampus and liver, compared to the germline. Hippocampus contains 5,494 piRNA-like peaks, the highest expression among all tested somatic tissues, followed by cortex (1,963), kidney (580), and liver (406). The study identifies 26 piRNA sequence species and 40 piRNA locations exclusive to all examined somatic tissues. Although piRNA expression has long been considered exclusive to the germline, our results support that piRNAs are expressed in several somatic tissues that may influence piRNA functions in the soma. Once confirmed, the PIWI/piRNA system may serve as a potential tool for future research in epigenome editing to improve human health by manipulating DNA methylation.

Keywords: DNA methylation; Mouse; epigenome editing; piRNA; piwi; somatic.

Figures

Figure 1.
Figure 1.
mRNA expression of piRNA machinery in mouse tissues. Quantitative RT-PCR analysis using the cDNA generated from three male (blue) and three female (red) sets of two-month-old adult brain, hippocampus, heart, kidney, liver, and testis/ovaries. The Ct values for each promoter was first normalized with an internal control (β-actin) to generate ΔCt and subsequently used for calculating gene expression levels (ΔΔCt). A range of average Ct values for each tissue is also presented below the graph to indicate the absolute expression levels of: (a) PIWIL1 mRNA expression (b) PIWIL2 mRNA expression and (c) PIWIL4 mRNA expression. The error bars represent the average from standard deviations calculated by triplicates.
Figure 2.
Figure 2.
Computational pipeline for small RNA sequencing analysis. Small RNAs (<200bp) were isolated from cortex (brain), hippocampus (brain), liver, kidney, and testis tissues from a pair of 2-month-old male mice. A subset of the small RNAs were treated with sodium periodate to identify 2ʹ-O-methylation signature, while the remaining untreated small RNA served as a control. All samples were library prepped, multi-plexed 10 per lane, and sequenced on an Illumina v4 HiSeq2500 with 50-bp single-end reads. The resulting sequence reads were first trimmed for adapters and size selected for 10–45 bp reads for quality control purposes (1Cutadpter). The sequences were then aligned to the mm10 mouse genome assembly (2STAR aligner) to filter out GENCOE exons, miRNAs, other small RNAs and tRNA species from analysis. The resulting reads were compared with periodate treated and control samples to identify peaks (3 Pepr peakcalling.pl), which were defined as piRNA-like transcripts. The piRNA expression levels (4BedTools multicov) and sequence motifs were generated, allowing for ±3bp flanking region for each tissue (5WebLogo). Somatic piRNA tissues were compared based on location, sequence composition, and relative expression. The total piRNA peaks/reads were compared with existing piRNA databases: piRBase, piRNAbank, piRNA cluster database, and piRNAQuest.
Figure 3.
Figure 3.
Somatic piRNA characteristics. (a) Schematic representation of a typical germline piRNA. The ‘N’ symbolizes an RNA nucleotide (A- adenine, G- guanine, C- cytosine, or U-uracil). The subscripts signify the nucleotide number to infer the length of a piRNA, which may range from 24-32bp as indicated by the bracketed area. Germline piRNA characteristics include 24-32bp in length shown in blue, 5ʹ uridine signature (red arrow), adenosine signature at the 10th position (red arrow), and a 2ʹ -O-methylation signature at the 3ʹ end (red arrow). (b) Length distribution of peaks identified by our computational pipeline. The y-axis of histogram is represented in a logarithmic scale. The yellow bars represent peaks in which length ≥20bp and were defined as piRNA-like transcripts in this study. The blue bars represent shorter peaks (<20bp), which were excluded from further analyses. (c) Sequence logos of somatic piRNA-like transcripts. The sequence motifs were generated using the first 20bp from the 5ʹ end of piRNA-like transcripts, allowing for ±3bp flanking region to determine the strand direction. The number of peaks indicating a uridine signature at the 5ʹ end (U1) and an adenine at the 10th position (A10) are shown directly above the sequence motifs to its corresponding tissue.
Figure 4.
Figure 4.
The piRNA transcripts represented by various repeat classes. The pie charts represent the distribution of repeats for both somatic piRNAs (cortex, hippocampus, kidney, and liver) and germline piRNAs (from testis) detected from periodate treated samples in two biological replicates. The RepeatMasker software was used to annotate the repeats. The number of bases from the piRNA transcripts that overlapped with repetitive regions (‘masked’ through RepeatMasker) at each tissue and the mouse genome (mm10) are represented as a percentage. The repeat classifications include short interspersed elements (SINE), long interspersed elements (LINE), long terminal repeat elements (LTR), DNA elements, small RNA elements, and unclassified elements. The normalization is relative to the mouse genome mm10 RepeatMasker information.
Figure 5.
Figure 5.
piRNA sequence comparisons between somatic and germline tissues. (a) Tissue-specific piRNA sequences. The four two-way Venn diagrams represent the unique piRNA sequence comparisons between testis (pink) and cortex (gray), hippocampus (blue), kidney [47], and liver (orange), respectively. (b) Somatic piRNA sequence comparison between the three germ layers. The four-way Venn diagram indicates the piRNA sequences that are unique to somatic tissues including the brain and hippocampus for ectoderm, kidney for mesoderm, and liver for endoderm. It also indicates the piRNA sequences that are common between two or more tissue types.
Figure 6.
Figure 6.
Surveys of public piRNA databases and periodate treated piRNA for brain tissues in the current study. (a) Comparisons of currently available piRNA databases. The four-way Venn diagram represents the piRNAs annotated by the four available piRNA databases: piRBase (10,211,139), piRNAbank (659,639), piRNA cluster database (29,349), piRNAQuest (3,646,281). (b) The cortex (grey) and hippocampus (blue) piRNA transcripts comparison with the four databases. Based on the database comparison, 1,629 of 1,963 and 3,330 of 5,494 piRNA transcripts were also annotated in at least one of the four databases for cortex and hippocampus, respectively. Each Venn diagram represents periodate treated samples derived from two-month-old adult mice.

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