Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice

Abstract

Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.

Keywords: DNA methylation; helicase; spermatogenesis.

Fig. 1.

TDRD12 associates with mouse secondary piRNA biogenesis factors. (A) Domain organization of TDRD12 proteins in mouse (Mm), Drosophila (Dm), and Bombyx (Bm). (B) Immunoprecipitated (IP) complexes were probed by Western analysis for the indicated proteins. +RNaseA, treatment with RNaseA. (C) Sequence alignment of tudor domain from proteins demonstrated to bind symmetrical dimethyl arginine (sDMA), and those from TDRD12 proteins. Aromatic cage residues essential for binding sDMA are highlighted in red. (D) The read-length distribution in small RNA libraries from indicated proteins.

Fig. 2.

Roles of Tdrd12 domains in Piwi interaction and nuage localization. (A) Domain organization of Bombyx Tdrd12 (BmTdrd12). An HA-tagged version lacking the N-terminal region (259 aa) was used for cell-culture studies (Experimental Procedures). Other deletion versions used in this study are indicated. (B) Immunoprecipitated (IP) endogenous BmTdrd12 associates with endogenous Piwi protein Siwi. (C) HA-tagged Siwi, but not HA-Ago3, associates with endogenous BmTdrd12. (D) The nucleotide bias for a uridine at first position (1U) or adenosine at 10th position (A10) of reads in small RNA libraries is shown. (E) Analysis of HA-BmTdrd12 deletion versions for association with endogenous Siwi. (F) Subcellular localization of HA-BmTdrd12 and its mutants. Alignment of helicase domains from indicated proteins showing critical residues required for ATP binding or hydrolysis. Residues mutated in HA-BmTdrd12 are highlighted in red. (G) Helicase domain mutations in HA-BmTdrd12 do not affect association with endogenous Siwi.

Fig. 3.

Tdrd12 mutant male mice are infertile and display derepression of retrotransposons. (A) The nonsense C-to-A nucleotide substitution in exon 8 of Tdrd12 in repro23 mice leads to truncation of the coding sequence. (B) Atrophied testes of homozygous (−/−) Tdrd12 mutants compared with their heterozygous (+/−) littermates. (C) Hematoxylin/eosin staining of adult testes sections from Tdrd12 animals. es, elongating spermatids; rs, round spermatids; sp, spermatocytes. (Scale bar: 20 µm.) (D) Northern analysis for LINE1 (L1) and IAP retrotransposons in Tdrd12 mutants. Only L1 elements are activated in Tdrd1 mutants. Loading control is provided by ethidium bromide staining of ribosomal RNA (rRNA). (E) Immunofluorescence detection of L1ORF1p in adult Tdrd12 mutant testis. (F) Promoter CpG DNA methylation (indicated as filled circles) on transposon promoters quantified (in percentages) by bisulfite sequencing.

Fig. 4.

TDRD12 is required for biogenesis of MIWI2-bound secondary piRNAs. (A) Read-length distribution profile of testes total small RNAs from indicated animals. (B) Normalized (to microRNAs) density of reads mapping to IAP transposon consensus from P0 total small RNA libraries. Note the reduction of antisense reads in the Tdrd12 mutant. (C) Ratio of antisense/sense piRNAs mapping LINE1 or IAP consensus. (D) Association of piRNAs with Piwi proteins examined by immunoprecipitation and 5′-end labeling from newborn pups (P0). RNA markers (nucleotides, nt) are shown. (E) Immunofluorescence detection of proteins in embryonic testes from indicated Tdrd12 genotypes. Images (green) are shown along with DAPI staining for DNA. (Scale: Width of each panel is 70 μm.) Note the loss of nuclear MIWI2 staining in Tdrd12 mutants. (F) Plot showing 5′-end overlap of opposing reads on IAP consensus in total small RNA libraries. Note the reduction in signal at position 10 (Ping-Pong signature) in the Tdrd12 mutant. (G) Current knowledge on factors implicated in the mouse piRNA pathway, and placement of TDRD12 as a secondary piRNA biogenesis factor.