MIWI N-terminal arginines orchestrate generation of functional pachytene piRNAs and spermiogenesis

Abstract

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.

Graphical Abstract

Figure 1.

Miwi^RK/RK mice are infertile due to spermatid elongation failure. (A) Schematic representation of wild-type (WT) and arginine to lysine (RK) mouse MIWI proteins. (B) Littersize per plug from Miwi^WT and Miwi^RK/RK studs mated to WT females and (C) Adult testis weight; P-value of two-tailed Student's t-test. (D) Representative images of PAS-stained testis and epididymis sections from Miwi^+/RK and Miwi^RK/RK; scale bar, 20 μm. (E) Spermatid elongation arrest at step 8–9. PAS-stained testis sections for different seminiferous cycle stages. PL, pre-leptotene; L, leptotene; Z, zygotene; eP, early pachytene; P, pachytene; lP, late pachytene; m2, secondary meiocyte; RS, round spermatid; eS (13), elongating spermatid (step 13). Scale bar, 5 μm. (F) Western blots from indicated proteins in P24 testis lysates; beta-tubulin (TUBB) serves as loading control.

Figure 2.

MIWI-NTRs sustain pachytene piRNA amplification of MIWI bound piRNAs and transposon control. (A, B) Western blots of MIWI immunoprecipitates from indicated P24 testis lysates. (C) Proteins (top) and bound piRNAs (bottom) of MILI and MIWI immunoprecipitates from indicated P24 testis lysates. (D) Electron micrographs of Miwi^+/RK and Miwi^RK/RK pachytene spermatocytes; Nu, nucleus; dashed line, nuclear membrane; Mi, mitochondrion; red arrowheads, intermitochondrial cement; scale bar, 400 nm. (E, F) 5′–5′ distance (ping-pong) analyses and Z-scores of MILI-bound (E) and MIWI-bound (F) piRNAs mapping to pachytene clusters in indicated genotypes. (G) Differential expression analysis of transposons between Miwi^+/RK and Miwi^RK/RK calculated from RNA-Seq libraries of P24 testes; top, MA plot (average expression relative to fold-change); bottom Volcano plot (statistical significance relative to fold-change); red, adjusted P-value <0.05 and log₂ fold-change ≥1; blue, adjusted P-value <0.05 and log₂ fold-change ⇐–1; grey, adjusted P-value ≥0.05 and/or log₂ fold-change > −1 < 1.

Figure 3.

MIWI-RK impacts the transcriptome but not the translatome. (A, B) Differential expression analysis calculated from RNA-Seq (A) and differential ribosome occupancy calculated from Ribo-seq (B) between Miwi^+/RK and Miwi^RK/RK P24 testes, visualized by Volcano (left) and MA (right) plots; red, adjusted P-value < 0.05 and log₂ fold-change ≥1; blue, adjusted P-value <0.05 and log₂ fold-change ⇐–1; grey, adjusted P-value ≥0.05 and/or log₂ fold-change > −1 < 1.

Figure 4.

MIWI-NTRs sustain piRNAs that cleave and destabilize select mRNAs essential for spermiogenesis. Metatranscript distribution of predicted, piRNA-mediated cleavages on mRNA targets, without (‘theoretical’, A), or with degradome-Seq support (B). (C) Sequence and genomic location of piRNAs found to cleave and destabilize the listed mRNAs at the indicated coordinates. piRNAs and cleaved RNA fragments map to the following categories (green boxes): Cl: piRNA cluster; S: sense-aligning repeat sequence; AS: antisense-aligning repeat sequence; E: exonic sequence. Arrows indicate changes in the expression of piRNAs (down) and mRNA targets (up) in Miwi^RK/RK compared to Miwi^+/RK.

Figure 5.

MIWI-NTRs are required for TDRD6 interaction, chromatoid body compaction and stability of key spermiogenic mRNAs. (A) Western blots of MIWI immunoprecipitates from indicated P24 testis lysates. (B) Immunofluorescence staining for MIWI (green) and TDRD6 (red) on round spermatid populations of Miwi^+/RK and Miwi^RK/RK P24 testis sections. Scale bar, 10 μm. (C) Electron micrographs of Miwi^+/RK and Miwi^RK/RK round spermatids; Nu, nucleus; red arrowheads, chromatoid body; scale bar, 400 nm. (D) pre-mRNA and mRNA relative expression levels for indicated genes, assayed by RT-qPCR (biological and technical triplicates) in Miwi^+/RK and Miwi^RK/RK P24 testes. Assays for Tcp1 were used to normalize for loading and differentiation stage; P-values of one-sided t-test.

Figure 6.

Model of MIWI-NTR functions. (A, B) MIWI-NTR function in piRNA amplification, biogenesis, transposon clearance and gene targeting (A), and in chromatoid body compaction and spermiogenic mRNA stability (B).