Widespread expression of piRNA-like molecules in somatic tissues

Abstract

Piwi-interacting RNA (piRNA) are small RNA abundant in the germline across animal species. In fruit flies and mice, piRNA have been implicated in maintenance of genomic integrity by transposable elements silencing. Outside of the germline, piRNA have only been found in fruit fly ovarian follicle cells. Previous studies have further reported presence of multiple piRNA-like small RNA (pilRNA) in fly heads and a small number of pilRNA have been reported in mouse tissues and in human NK cells. Here, we analyze high-throughput small RNA sequencing data in more than 130 fruit fly, mouse and rhesus macaque samples. The results show widespread presence of pilRNA, displaying all known characteristics of piRNA in multiple somatic tissues of these three species. In mouse pancreas and macaque epididymis, pilRNA abundance was compatible with piRNA abundance in the germline. Using in situ hybridizations, we further demonstrate pilRNA co-localization with mRNA expression of Piwi-family genes in all macaque tissues. Further, using western blot, we have shown the expression of Miwi protein in mouse pancreas. These findings indicate that piRNA-like molecules might play important roles outside of the germline.

Figure 1.

piRNA and pilRNA features in fly tissues. (A) Length distribution of all mapped sequence reads in fly tissues. Here (and further), the labels indicate: OSS, ovarian somatic sheet cells; EMB, embryo; OVY, ovary; HED, head; IMD, imaginal discs. For a full sample description, see Supplementary Table S13. (B) Length distribution of all mapped sequence tags (sequence reads after collapsing identical sequences). (C and D) Length distributions of sequence reads and tags, excluding reads mapped to known ncRNA, respectively. Note that 5′ uridine bias is one known property of primary piRNA. (E) Proportions of complementary small RNA reads (24–32 nt) at different offsets. Excess of complementary reads at offset equal 10 nt indicates the ping-pong model signature. (F) The stand (S) and the 5′ uridine (U) bias of sequence reads' clusters based on 24–32 nt long sequences. (G) The upper panel shows genomic context of small RNA clusters (24–32 nt) identified in five fly tissues using publicly available data sets (11,21,29,30–33). The lower panel shows relative abundance of uniquely mapped small RNA (24–32 nt) among four previously characterized fly TE master loci (11).

Figure 2.

piRNA and pilRNA features in mouse tissues. (A) Length distribution of all mapped sequence reads in three adult mouse tissues: Te, testes; Ov, ovary; Pa, pancreas. (B) Length distribution of all mapped sequence tags (sequence reads after collapsing identical sequences). (C and D) Length distributions of sequence reads and tags, excluding reads mapped to known ncRNA, respectively. Note that 5′ uridine bias is one known property of primary piRNA. (E) Proportions of complementary small RNA reads (24–32 nt) at different offsets. Excess of complementary reads at offset equal 10 nt indicates the ping-pong model signature. (F) The stand (S) and the 5′ uridine (U) bias of sequence reads' clusters based on 24–32 nt long sequences. (G) Reads coverage of a bidirectional piRNA cluster previously identified in mouse testes (4) in three mouse tissues. Shown are all reads with length 24–32 nt, excluding reads mapped to known ncRNA. The y-axis shows log2-transformed nucleotide coverage normalized by the total number of mapped reads within in a given sample. (H) Genomic context of piRNA clusters found in mouse testes at different age, from embryonic stages, pre-pachytene (10 dpp) and pachytene (18 dpp) stages to adults, based on data from (7,10,16,26,27) (‘Materials and Methods' section). The labels indicate the age of the samples in days post-conception (dpc), days post-partum (dpp) or weeks (w) and piRNA origin: testes tissue (no label) or immunoprecipitations of Mili (Ml), Miwi2 (Mw2) or Miwi (Mw) proteins. For a full sample description, see Supplementary Table S13. (I) Genomic context of pilRNA clusters in adult mouse tissues: GC-B, germinal center B cells; Ma-B, mature B cells; Pl, plasma cells; FH-T, follicular helper T cells; NK, natural killer cells; MP, macrophages; MDD, macrophages-derived dendritic cells; Ne, neutrophils; HP, hematopoietic progenitor cells; Ov, ovaries; Pa, pancreas; Te, testes (28).

Figure 3.

piRNA and pilRNA features in rhesus macaque tissues. (A) Length distribution of all mapped sequence reads in five adult rhesus macaque tissues and adult mouse testes. The labels indicate: R.Te, macaque testes; R.Ep, macaque epididymis; R.Pr, macaque prostate; R.Sv, macaque seminal vesicles; R. Co, macaque cerebral cortex; M.Te.All, mouse total testes (26); M.Te.ML, mouse testes Mili-immunoprecipitation (16); M.Te.MW, mouse testes Miwi-immunoprecipitation (16) (B) Length distribution of all mapped sequence tags (sequence reads after collapsing identical sequences). (C and D) Length distributions of sequence reads and tags, excluding reads mapped to known ncRNA, respectively. Note that 5′ uridine bias is one known property of primary piRNA. (E) Proportions of complementary small RNA reads (24–32 nt) at different offsets. Excess of complementary reads at offset equal 10 nt indicates the ping-pong model signature. (F) The stand (S) and the 5′ uridine (U) bias of sequence reads' clusters based on 24–32 nt long sequences. (G) Reads coverage of an example piRNA with high coverage in adult macaque testes. Shown are all reads with length 24–32 nt, excluding reads mapped to known ncRNA. The y-axis shows log2-transformed nucleotide coverage normalized by the total number of mapped reads within in a given sample. The bottom of the panel shows annotation of the corresponding rhesus macaque genomic region, dark green—SINE and location of one of the ‘anti-pilRNA’ LNA probes (piRNAa)—arrow. Note that the genomic region orthologous to the macaque small RNA cluster shown in Figure 3G is inverted in the mouse genome. To align the mouse and macaque genomic regions, mouse cluster is shown in reverse orientation with respect to the macaque cluster.

Figure 4.

Localization of pilRNA and Piwi-family mRNA expression in macaque tissues. (A) In situ hybridizations with two different ‘anti-pilRNA’ LNA probes (piRNAa and piRNAb) and probes against four rhesus macaque PIWI transcripts (PIWIL1-4) in adult rhesus macaque tissues. Negative control shows tissue staining after identical hybridization and staining procedure, omitting specific probes. (B) Rhesus macaque epididymis tissue before and after LCM of principal/basal cells (pilRNA stained) and peritubular tissue (pilRNA free). (C) Small RNA sequencing coverage of pilRNA clusters (red) and background expression clusters (gray) (‘Materials and Methods' section) in rhesus macaque LCM-purified principal/basal cells (pilRNA stained) and peritubular tissue (pilRNA free).