PNLDC1 is essential for piRNA 3' end trimming and transposon silencing during spermatogenesis in mice

Abstract

Piwi-interacting RNAs are small regulatory RNAs with key roles in transposon silencing and regulation of gametogenesis. The production of mature piwi-interacting RNAs requires a critical step of trimming piwi-interacting RNA intermediates to achieve optimally sized piwi-interacting RNAs. The poly(A)-specific ribonuclease family deadenylase PNLDC1 is implicated in piwi-interacting RNA trimming in silkworms. The physiological function of PNLDC1 in mammals remains unknown. Using Pnldc1-deficient mice, here we show that PNLDC1 is required for piwi-interacting RNA biogenesis, transposon silencing, and spermatogenesis. Pnldc1 mutation in mice inhibits piwi-interacting RNA trimming and causes accumulation of untrimmed piwi-interacting RNA intermediates with 3' end extension, leading to severe reduction of mature piwi-interacting RNAs in the testis. Pnldc1 mutant mice exhibit disrupted LINE1 retrotransposon silencing and defect in spermiogenesis. Together, these results define PNLDC1 as a mammalian piwi-interacting RNA biogenesis factor that protects the germline genome and ensures normal sperm production in mice.piRNAs are regulatory RNAs that play a critical role in transposon silencing and gametogenesis. Here, the authors provide evidence that mammalian PNLDC1 is a regulator of piRNA biogenesis, transposon silencing and spermatogenesis, protecting the germline genome in mice.

Fig. 1

Pnldc1 is essential for spermatogenesis. a Testicular atrophy in Pnldc1 ^Mut mice. Testis sizes and weights of adult wild-type (WT) and Pnldc1 ^Mut mice are shown. n = 5; significance determined by unpaired Student’s t-test; **p < 0.01. Error bars represent s.e.m. b Spermiogenic arrest in adult Pnldc1 ^Mut mice. Hematoxylin and eosin-stained testis sections from adult WT and Pnldc1 ^Mut (Mut-5) mice are shown. AES abnormal elongated spermatids, P pachytene spermatocytes, R round spermatids. Scale bars, 100 μm (left) and 20 μm (right). c Hematoxylin and eosin-stained epididymis sections from adult WT and Pnldc1 ^Mut (Mut-5) mice are shown. Scale bar, 20 μm. d Meiotic arrest in adult Tdrkh KO mice. Hematoxylin and eosin-stained testis sections from adult WT and Tdrkh KO mice are shown. Z zygotene spermatocytes. Scale bar, 20 μm. e Co-immunostaining of CRISP2 (red) and γH2AX (green) in adult WT and Pnldc1 ^Mut (Mut-1) testes. DNA (blue) is stained by DAPI. Spermatogenic stages are noted. Scale bar, 20 μm. f The timeline of mouse spermatogenesis with red crosses representing the arrested spermatogenic stages in Pnldc1 ^Mut and Tdrkh KO testes

Fig. 2

Retrotransposon LINE1 derepression in Pnldc1 mutant spermatocytes. a In situ hybridization of LINE1 Orf1 mRNA in adult WT and Pnldc1 ^Mut (Mut-1) testes. LINE1 Orf1 mRNA was upregulated in spermatocytes in Pnldc1 ^Mut testes but was undetectable in WT testes. Scale bar, 50 μm. b Immunostaining was performed using LINE1 ORF1 antibody on adult testis sections from WT and Pnldc1 ^Mut (Mut-1) mice. LINE1 ORF1 was upregulated in spermatocytes in Pnldc1 ^Mut testes but was undetectable in WT testes. Scale bar, 20 μm. c Immunostaining was performed using LINE1 ORF1 antibody and γH2AX antibody on adult testis sections from WT and Pnldc1 ^Mut (Mut-1) mice. Different cell types were distinguished according to γH2AX staining and DAPI staining. LINE1 ORF1 was expressed from zygotene spermatocytes to mid-pachytene spermatocytes in Pnldc1 ^Mut testes. Scale bar, 5 μm

Fig. 3

Pnldc1 mutation causes mitochondria disorder. a Expression of TDRKH, MILI, MIWI, GASZ, and MVH in WT and Pnldc1 ^Mut (Mut-1) testes revealed by western blotting. β-actin is a loading control. b Aggregation of TDRKH, MILI, MIWI, and GASZ in Pnldc1 ^Mut spermatocytes. Immunostaining was performed using indicated antibodies on adult testis sections from WT and Pnldc1 ^Mut (Mut-1) mice. DNA (blue) is stained with DAPI. Protein aggregations are indicated by arrows. Scale bar, 10 μm. c Conglomeration of mitochondria in Pnldc1 ^Mut testes. Immunostaining of WT and Pnldc1 ^Mut (Mut-1) testis sections with an antibody against AIF, a mitochondrial marker. DNA (blue) is stained with DAPI. Conglomeration of mitochondria is indicated by arrows. Scale bar, 10 μm

Fig. 4

Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 ^Mut testes. a piRNA extension in Pnldc1 ^Mut mice. Total RNAs from adult WT and Pnldc1 ^Mut (Mut-1 and Mut-2) testes were end-labeled with [³²P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 ^Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [³²P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 ^Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [³²P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 ^Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 ^Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 ^Mut (Mut-1 and Mut-2) MIWI-piRNA libraries

Fig. 5

piRNA 3′ end extension in adult Pnldc1 ^Mut testes. a Genomic annotation of total piRNA, MILI-piRNAs, and MIWI-piRNAs from adult WT and Pnldc1 ^Mut (Mut-1) testes. Sequence reads (24–48 nt) from small RNA libraries were aligned to mouse sequence sets in the following order: piRNA clusters, coding RNA, non-coding RNA, repeats, and intronic sequences (see Methods for details). “Other” represents sequence reads that did not mapped to the above five sequence sets. The percentage of mapped reads is shown. b Nucleotide distributions at the first position in total piRNA, MILI-piRNAs, and MIWI-piRNAs from adult WT and Pnldc1 ^Mut (Mut-1) testes. The 24–40 nt reads from small RNA libraries were used. c Extended piRNA 3′ ends in Pnldc1 ^Mut testes. The 33–40 nt reads from Pnldc1 ^Mut (Mut-1 and Mut-2) total piRNA library were mapped to 24–32 nt reads from WT total piRNA library. The number of 33–40 nt Pnldc1 ^Mut piRNA reads that perfectly match at least one WT piRNA was calculated. The percentage of matching pairs that have identical 5′ ends or identical 3′ ends against all matched pairs are shown. n = 2. Error bars represent s.e.m. d Two examples of 3′ end extension in Pnldc1 ^Mut (Mut-1) piRNAs. Alignments between 33–48 nt reads from Pnldc1 ^Mut total piRNA library and 24–32 nt reads from WT total piRNA library within a selected region from two representative piRNA clusters. The genomic locations of these two piRNA clusters are shown at the bottom

Fig. 6

Pachytene piRNA biogenesis is phased in mice. a U bias at position +1 downstream of piRNA 3′ ends in Pnldc1 ^Mut total piRNA. The 24–48 nt reads from WT and Pnldc1 ^Mut (Mut-1) total piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1, the most abundantly expressed). Sequence logos showing nucleotide composition at mapped piRNA 5′ ends, 3′ ends, and downstream regions of 3′ ends were generated. Gray shading marks the piRNA region. b U bias at position +1 downstream of piRNA 3′ end in Pnldc1 ^Mut MILI-piRNAs and MIWI-piRNAs. The 24–48 nt reads from WT and Pnldc1 ^Mut (Mut-1) MILI- and MIWI-piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1). Sequence logos showing nucleotide composition in the vicinity of mapped piRNA 3′ ends were generated. Gray shading marks the piRNA region. c Untrimmed Pnldc1 ^Mut piRNAs exhibit coupling of piRNA 3′ ends with subsequent piRNA 5′ ends. The 24–48 nt reads from WT and Pnldc1 ^Mut (Mut-1) total piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1). Top 1000 distinctively mapped piRNAs were extracted as references to perform 3′–5′ coupling analysis. The frequency of piRNA 5′ ends around referenced piRNA 3′ ends was calculated. Z-scores at position +1 (Z1) are shown

Fig. 7

PNLDC1 is required for pre-pachytene piRNA trimming. a Immunostaining of MILI on newborn (P0) testis sections from Pnldc1 HET and Pnldc1 KO mice. Scale bar, 10 μm. b Immunostaining of MIWI2 on testis sections from Pnldc1 HET and Pnldc1 KO mice. MIWI2 is localized in both cytoplasm and nucleus in P0 Pnldc1 KO testes. Scale bar, 10 μm. c Immunostaining of MIWI2 on P0 testis sections from Tdrkh HET and Tdrkh KO mice. MIWI2 exclusively localizes in the cytoplasm of P0 Tdrkh KO germ cells. Scale bar, 10 μm. d The length distribution of MILI-piRNAs from P0 Pnldc1 HET and Pnldc1 KO MILI-piRNA libraries. e piRNA 3′ end extension in P0 Pnldc1 KO mice. Alignment of 31–40 nt reads from P0 Pnldc1 KO MILI-piRNA library and 24–30 nt reads from P0 Pnldc1 HET MILI-piRNA library within a selected region in one representative piRNA cluster. The genomic location of this piRNA cluster is shown at the bottom