Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline

Abstract

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1'U-containing, 2'O-methylated 31-37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3'→5' processing of 31-37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.

Figure 1

Tdrkh interacts with Miwi and Miwi2. (A) FLAG-tagged Miwi and myc-tagged Tdrd proteins were co-transfected into 293T cells followed by immunoprecipitation of the myc-tagged proteins. Tdrkh is also known as Tdrd2. (B) Myc-tagged Tdrkh was co-transfected with FLAG-tagged Piwi and Ago proteins into 293T cells, followed by immunoprecipitation of the FLAG tag. (C) Co-immunoprecipitation of Miwi and Mili followed by western blot detection of Tdrkh from 2-month-old CD-1 mouse testis extract. (D) Co-immunoprecipitation of Miwi by Tdrkh from 2-month-old CD-1 mouse testis extract treated with or without RNase A. (E) Domain mapping of the Miwi–Tdrkh interaction by co-transfection with FLAG-tagged Miwi truncations and myc-Tdrkh. (F) Domain mapping of the Miwi–Tdrkh interaction by co-transfection with myc-tagged Tdrkh truncations and FLAG-Miwi. Arrows indicate specific western blot bands. (G) Co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi2 containing point mutations to prevent symmetric dimethylation. (H) Co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi containing point mutations to prevent symmetric dimethylation. (I) siRNA-mediated knockdown of PRMT5 prevents co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi and FLAG-Miwi2. (J) Biotinylated Miwi peptides corresponding to Miwi residues 2–17, either with no methylated residues (Miwi2–17) or with symmetric dimethylation on arginine 4 (R4(me₂s)) or arginine 14 (R14(me₂s)), were incubated with adult C57 testis lysate prepared with 150 mM salt followed by western blot analysis for Tdrkh.

Figure 2

Tdrkh is required for male fertility. (A) Western blot analysis of tdrkh +/+, +/−, and −/− animal testis lysate. (B) Two-month-old testis from tdrkh +/− and −/− animals, ticks=0.1″. (C) H&E staining of 2-month-old +/− and −/− testis sections, scale bar=50 μm. (D) Quantitation of 14dpp spermatocyte spreads, N(+/−)=1821, N(−/−)=848. (E) Labelling of spermatocyte spreads from 14dpp +/− and −/− testis stained with Sycp1 and Sycp3. L, leptotene; Z, zygotene; P, pachytene, scale bar=10 μm. (F) Double labelling of 14dpp spermatocyte spreads with Rad51 (red) and Sycp3 (green). L, leptotene; Z, zygotene; Z/P, zygotene/pachytene; P, pachytene. Scale bar=10 μm. (G) Double labelling of 14dpp spermatocyte spreads with γH2A.X (red) and Sycp3 (green). L, leptotene; Z, zygotene; P, pachytene, scale bar=10 μm. (H) Staining of 14dpp germ cell spreads. Spermatogonia stained with anti-phospho-S25 53BP1, spermatocytes stained with total anti-53BP1, scale bar=50 μm. (I) Staining of 2-month-old CD-1 testis sections with anti-phospho-S25 53BP1 (green) and laminin (red), scale bar=75 μm. (J) TUNEL labelling (green) of 2-month-old +/− and −/− testis sections, scale bar=75 μm. (K) Western blot analysis of 11dpp Tdrkh +/− and −/− lysates. C, UV-irradiated CCE ES cell extract.

Figure 3

Tdrkh represses Line1 expression. (A) Localization of pi-body (Mili, Tdrd1), piP-body (Miwi2, Mael), and pan-nuage (Mvh) components in 18dpc tdrkh +/− and −/− testis sections, scale bar=10 μm. (B) Electron microscopy of 11dpp tdrkh +/− and −/− spermatocytes. Arrowheads indicate nuage/intermitochondrial cement. Scale bar=300 nm. (C) RT-qPCR analysis of transposon expression in three independent pairs of 12dpp testis for each genotype. Error bars indicate standard deviation. (D) Line1 Orf1p expression (green) in 18dpc, 7dpp, 11dpp, and adult tdrkh +/− and −/− testis sections. Scale bar=75 μm.

Figure 4

Tdrkh functions in primary piRNA biogenesis. (A) Size distribution of small RNA libraries from 18dpc tdrkh +/− and −/− testes, without normalization. (B) Ratio of 18dpc piRNA levels in Tdrkh mutant testes derived from the top 19 embryonic piRNA clusters, relative to tdrkh +/− levels. (C) Percentage of total library reads mapping to indicated categories in 18dpc libraries. (D) Percentage of repeat-associated reads mapping to indicated transposon family in control (upper) and mutant (lower) 18dpc libraries. (E) Ratio of sense and antisense 18dpc piRNAs mapping to specified transposons, relative to control levels. (F) Ratio of 18dpc piRNA reads mapping to indicated transposon families, relative to control levels. (G) Levels of piRNAs mapping to indicated mRNA region, expressed as a percentage of total reads in each genotype. (H) Distances between 5′ complementary ends of piRNA mapping to IAP consensus sequence were measured to calculate a ping-pong biogenesis signature. (A–H) 18dpc libraries. (I) Size distribution of small RNA libraries from 11dpp tdrkh +/− and −/− testes, without normalization. (J) Ratio of 11dpp piRNA levels in Tdrkh mutant testes derived from the top 19 post-natal pre-pachytene piRNA clusters, relative to control reads. (K) Percentage of total library reads mapping to indicated categories in 11dpp libraries. (L) Percentage of repeat-associated reads mapping to indicated transposon family in control (upper) and mutant (lower) 11dpp libraries. (M) Ratio of sense and antisense 11dpp piRNAs mapping to specified transposons, relative to control levels. (N) Ratio of 11dpp piRNA reads mapping to indicated transposon families, relative to control levels. (O) Levels of 11dpp piRNAs mapping to indicated mRNA region, expressed as a percentage of total reads in each genotype. (I–O) 11dpp libraries.

Figure 5

Tdrkh facilitates piRNA maturation from 31 to 36 nt Mili-bound piRNA intermediates. (A) End labelling and small RNA analysis of Mili-bound small RNAs in 11dpp tdrkh^+/− and tdrkh^−/− testes reveals the accumulation of piRNA intermediates in the mutant testes. (B) Size distribution of Mili-associated RNA libraries from 11dpp tdrkh^+/− and tdrkh^−/− testes, without normalization. (C) Nucleotide distribution at the first position in 24–31 nt and 32–40 nt Mili-associated RNAs from tdrkh^+/− and tdrkh^−/− testes. (D, E) Alignments between mature and precursor piRNAs within a 1-kb window of piRNA clusters on chromosome 10 (D) and chromosome 8 (E). All mature piRNAs that perfectly map to an intermediate are shown, and the alignment of the pair is presented in the plots. Note that all alignments posses perfectly aligned 5′ ends. Mature piRNA=black, intermediate=red. (F) Northern blot analysis of total 11dpp RNA using an antisense probe that recognizes a mature 27-nt Line1 piRNA (upper panel), an antisense probe that recognizes its 31 bp intermediate form (middle), or U6 RNA as a loading control (bottom). (G) RNase protection assay using total 11dpp RNA and a radiolabelled probe that can be protected from RNase digestion by both the mature 27 nt Line1 piRNA and its 31nt intermediate form. * indicates non-digested probe, arrowhead indicates the intermediate piRNA, and the arrow indicates the mature piRNA. (−) in the RNA lane indicates only yeast RNA was present in the hybridization. +/− and −/− indicate genotypes of Tdrkh animals. (H) Northern blot analysis of total 11dpp RNA for expression of a cluster-derived piRNA using an antisense probe, which recognizes the mature and intermediate forms of the piRNA (upper panel), a longer antisense probe that specifically recognizes only the intermediate form (middle panel), or an antisense probe for U6 as loading control (middle, bottom panels). (I) High-resolution northern blotting of total 11dpp +/− and −/− RNA for expression of a cluster-derived piRNA and its intermediate, using the same antisense probe in (H). The mature form is visible in the +/− sample while the intermediate is present in the −/− sample. (J, K) High-resolution northern blot analysis following treatment of total mutant 11dpp RNA with water (C) or with NaIO₄ and β-elimination (β). Non-protected 3′ ends will result in RNAs which migrate ∼2 nt faster than control reactions. M, 1nt marker. (J) Northern blot with an antisense probe for the cluster-derived piRNA intermediate studied in (H, I). (K) Northern blot with an antisense probe for the let-7c miRNA. (L) Abundance of 24–31 nt and 32–40 nt Mili-associated intermediate RNA levels in tdrkh^−/− testes derived from the top 19 post-natal pre-pachytene piRNA clusters, normalized to 24–31 nt control reads. (M) Ratio of mature (27 nt) and intermediate (31–36 nt) Mili-associated piRNAs between tdrkh^+/− and tdrkh^−/− testes mapped to L1MdGf, L1MdA, and IAP retrotransposons. (N) Ratio of mature (27nt) and intermediate (31–36nt) Mili-associated piRNAs from tdrkh^+/− and tdrkh^−/− testes mapped to introns, 5′UTRs, coding regions, and 3′UTRs. (O) Percentage of total library reads that are mapped to indicated categories in tdrkh^+/− (upper) and tdrkh^−/− (lower) libraries. (P) Percentage of repeat-associated reads mapped to indicated categories in tdrkh^+/− (upper) and tdrkh^−/− (lower) libraries.

Figure 6

A model of Tdrkh function in piRNA biogenesis and the Piwi-piRNA pathway. Mili-associated piRNA precursors (red line) are selected and loaded into Mili complexes and processed into intermediates. Tdrkh recruits or facilitates activity of a 3′ exonuclease, ‘trimmer’, in the nuage, which proceeds to trim the intermediates to mature piRNA length. In addition, in embryonic testes, Tdrkh may function in transferring mature piRNAs or piRNA intermediates between Mili and Miwi2 complexes. Mili- and Miwi2-piRNA complexes proceed to enact epigenetic regulation, transposon (Tn) repression, translational regulation, and possibly surveillance of genomic integrity. In response to indefensible attacks on genomic integrity, Tdrkh may function in sensing or transducing apoptotic responses to mitochondria. See Discussion for details.