Two modes of targeting transposable elements by piRNA pathway in human testis

Abstract

PIWI proteins and their partner small RNAs, termed piRNAs, are known to control transposable elements (TEs) in the germline. Here, we provide evidence that in humans this control is exerted in two different modes. On the one hand, production of piRNAs specifically targeting evolutionarily youngest TEs (L1HS, L1PA2-L1PA6, LTR12C, SVA) is present both at prenatal and postnatal stages of spermatogenesis and is performed without involvement of piRNA clusters. On the other hand, at postnatal stages, piRNAs deriving from pachytene clusters target "older" TEs and thus complement cluster-independent piRNA production to achieve relevant targeting of virtually all TEs expressed in postnatal testis. We also find that converging transcription of antisense-oriented genes contributes to the origin of genic postnatal prepachytene clusters. Finally, while a fraction of pachytene piRNAs was previously shown to arise from long intergenic noncoding RNAs (lincRNAs, i.e., pachytene piRNA cluster primary transcripts), we ascertain that these are a specific set of lincRNAs that both possess distinguishing epigenetic features and are expressed exclusively in testis.

Keywords: PIWI proteins; piRNA cluster; piRNA pathway; transposable elements.

FIGURE 1.

piRNAs deriving from transposable elements (TEs) in postnatal testis. (A) Relationship between expression level of a TE and amount of all piRNAs deriving from it. (B) Relationship between genomic fractions of TE subfamilies in pachytene clusters and in randomized intergenic controls demonstrates enrichment of pachytene piRNA clusters with LTRs and depletion of LINE elements from them. TE subfamilies not presented in randomized controls or pachytene clusters are shown to the left of the y-axis and below the x-axis, respectively. (C) Relationship between expression level of a TE and amount of piRNAs deriving from pachytene clusters only. TE subfamilies without reads deriving from pachytene clusters are shown below the x-axis. All results are presented for pachytene clusters constructed with empirical approach (averaged across all postnatal testis small RNA-seq data sets under study and three postnatal testis RNA-seq data sets).

FIGURE 2.

Production of piRNAs deriving from transposable elements (TEs) in prenatal testis. (A) Top 30 TE subfamilies producing most piRNAs sorted in descending order for library F1 (prenatal testis, nonoxidized). (B) Distribution of piRNA reads along L1HS consensus in prenatal (F1, nonoxidized; FO1–FO2, oxidized) and postnatal testis libraries (A1–A6, nonoxidized). Scale ranges in RPM (reads per million) are given in brackets.

FIGURE 3.

piRNA production from 3′UTRs is associated with converging transcription of genes (transcription of antisense-oriented 3′UTRs). (A) Examples of converging transcription of 3′UTRs accompanied by significant piRNA production. Genome coordinates (assembly hg38) and a separate track for 21- to 23-nt reads (putative endo-siRNAs) are shown. (B) Fractions of antisense oriented genes adjacent to 1/3/5% of top piRNA-producing 3′UTRs. Top piRNA-producing 3′UTRs were defined based on the density of piRNAs per kilobase. Controls are random size-matched sets of 3′UTRs. (C) Median distance to antisense-oriented genes adjacent to 1/3/5% of top piRNA-producing 3′UTRs. Top piRNA-producing 3′UTRs and controls were defined as in panel B. The graphs in panels B and C show median, interquartile range (box), and min/max range (whiskers) across 11 postnatal libraries. P-value for two-tailed Mann–Whitney test is presented.

FIGURE 4.

Expression of “piRNA-producing lincRNA” is mostly restricted to testis. Fraction of testis-specific lincRNAs among all lincRNAs, testis-expressed lincRNAs, and “piRNA-producing lincRNAs” selected with a threshold of 1 RPKM or 10 RPKM. The graph shows median, interquartile range (box), and min/max range (whiskers) across either 100 randomly sampled sets of lincRNAs (for all/testis-expressed lincRNAs from GENOCODE v.23) or 11 postnatal libraries (for 1RPKM and 10RPKM piRNA-producing lincRNAs). P-value for two-tailed Mann–Whitney test is presented.