A novel class of somatic small RNAs similar to germ cell pachytene PIWI-interacting small RNAs

Abstract

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Keywords: Gene Regulation; RNA; RNA Interference (RNAi); Somatic Cell Genetics; Transposable Element (TE).

FIGURE 1.

Abundance and size of pilRNAs in Sertoli cells, ICCs, small intestine (Sm Int), and stomach. A, abundance of pilRNAs in small noncoding RNA 454 libraries of murine Sertoli cells purified from postnatal day 6 testes, ICCs purified from the gastrointestinal tracts of adult mice, and small intestine from adult mice. B, length distribution of all pilRNAs identified from the three 454 sncRNA libraries of somatic cells/tissue. The dominant size is at 30 nt, which is consistent with that of MIWI-associated/pachytene piRNAs. C, abundance of pilRNAs in small noncoding RNA PGM libraries of murine adult stomach. D, length distribution of all pilRNAs identified from stomach PGM sncRNA libraries. The dominant size is at 30 nt, which is consistent with that of MIWI-associated/pachytene piRNAs. E, scatter plots depicting log₂(normalized value + 1) expression between biological replicates from adult mice stomach small RNA. R² values were at least 0.98, indicating good correlation between biological replicates. Samples had an average depth of 4.4 × 10⁶ aligned reads.

FIGURE 2.

Validation of pilRNA expression in multiple murine tissues. Levels of three pilRNAs (pilRNA-in3, pilRNA-in18, and pilRNA-in87) identified from the small intestine sncRNA libraries and two germ cell piRNAs (piR-118029 and piR-126541) were determined using qPCR in 10 tissues including nine somatic tissues and the testis. U6 snRNA was used as an endogenous control, and all somatic tissue values are relative to levels of testis expression.

FIGURE 3.

Somatic pilRNAs resemble germ cell pachytene piRNAs. A, somatic cell-expressed pilRNAs identified by 454 sequencing display a preference for 5′ uracil but lack enrichment of an adenine at the 10^th position. B, PGM sequenced stomach pilRNAs display a preference for 5′ uracil but lack enrichment of an adenine at the 10^th position. C, clustering analyses of pilRNAs by aligning pilRNAs to known prepachytene and pachytene piRNA clusters. The number of pilRNAs found within one or both of the two known types of piRNA clusters is indicated. The majority of pilRNAs appear to belong to pachytene piRNA clusters. D, clustering analyses of pilRNAs by aligning pilRNAs to recently redefined prepachytene, hybrid, and pachytene piRNA clusters. The number of pilRNAs found within each category of piRNA clusters is indicated. The majority of stomach pilRNAs belong to pachytene piRNA clusters. Nt, nucleotide; Sm Int, small intestine.

FIGURE 4.

Nucleotide preference of pilRNAs that align to known piRNA and novel pilRNAs. A, ICC pilRNAs show a 5′ uracil preference and no 10^th nt preference in both groups. B, Sertoli pilRNAs that align known piRNAs show a 5′ uracil preference for both groups but less so in novel pilRNAs. Both groups show no 10^th nt preference. C, small intestine pilRNAs show a 5′ uracil preference and no 10^th nt preference in both groups. Nt, nucleotide; Sm Int, small intestine.

FIGURE 5.

Examples of stomach pilRNA clusters aligned to germ cell pachytene spermatocyte piRNA clusters. A, genomic view of pachytene spermatocyte and stomach reads aligned to intergenic bidirectional clusters 17-qA3.3–27363 and 17-qA3.3–26735. B, length histogram of stomach pilRNAs originating from clusters 17-qA3.3–27363 and 17-qA3.3–26735. Predominant size of stomach reads aligned to these two clusters is 30 nt, consistent with pachytene piRNAs. C, nucleotide composition of stomach pilRNAs originating from clusters 17-qA3.3–27363 and 17-qA3.3–26735. Stomach pilRNAs have a preference for 5′ uracil indicative of primary processing. D, precursor transcript RT-PCR. Precursor transcripts for germ cell piRNA clusters 2-qE1–35981, 9-qC-31469, 17-qA3.3–27363, and 17-qA3.3–26735 were detected in both stomach (st columns) and testis (tes columns) by two primer sets (Table 2). RNA was either treated with DNase (D columns) or RNase (R columns) as a negative control. All amplicons matched to their anticipated sizes (Table 2).

FIGURE 6.

Although pilRNAs are predominantly like germ cell pachytene piRNAs, pilRNAs still have ping-pong activity. A, results of pairwise alignments of 5′ 10- and 11-nucleotide overlaps in stomach pilRNAs. B, nucleotide composition of ping-pong stomach pilRNAs with 10-nucleotide 5′ overlaps. C, cluster alignments of ping-pong stomach pilRNAs with ping-pong signature. D, nucleotide composition of ping-pong stomach pilRNAs based on cluster classification.

FIGURE 7.

pilRNA production is independent of the known miRNA/endo-siRNA or piRNA biogenetic pathways. A, heat map representing the pilRNA transcriptome in wild-type, Dicer- or Drosha-null murine Sertoli cells based on small RNA sequencing. Expression is based on a Log₁₀ scale. B, scatter plots depicting log₂(normalized value) expression between replicates from FACS-purified Sertoli cells of Amh-cre control, Dicer conditional knock-out, and Drosha conditional knock-out mice. R² values were at least 0.82, indicating relatively good correlation between replicates. Samples had an average depth of ∼450,000 aligned reads. C, heat maps representing qPCR analyses of expression levels of three intestinal pilRNAs (pilRNA-in3, pilRNA-in18, and pilRNA-in87) and two known germ cell piRNAs (piR-118029 and piR-126541) in 10 organs of MIWI, MIWI2, and MOV10L1 global KO mice. U6 snRNA was used as an endogenous control, and values in KO samples were relative to respective values of wild-type samples.

FIGURE 8.

Partial complementary alignment of 696 pilRNAs identified in 454 sequencing to all known mRNAs. A, number of pilRNAs potentially targeting 3′-UTRs, 5′-UTRs, and CDSs of all known murine mRNAs. Shown are the results of reverse complementary match of pilRNAs to mRNAs when one to two or one to five mismatches were allowed. B, number of mRNA transcripts potentially targeted within the 3′-UTRs, 5′-UTRs, and CDSs by pilRNAs. Sm Int, small intestine.