The NMD endonuclease SMG6 co-operates with the piRNA pathway in germ granules to shape the male germ cell transcriptome

Nonsense-mediated RNA decay (NMD) is a conserved RNA turnover pathway. Here we report that the sole endonuclease in the NMD pathway, SMG6, is essential for the male germline. Germ-cell conditional knockout (cKO) of Smg6 causes complete arrest of spermatogenesis at the early haploid cell stage. Smg6-cKO round spermatids accumulate NMD target mRNAs with long 3' untranslated regions (UTRs) and fail to eliminate transcripts normally expressed during meiosis, the previous step in spermatogenesis. SMG6 and PIWI-interacting (pi) RNA pathway components are highly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells. This led to the intriguing possibility that the CB is a site where SMG6 and the piRNA pathway co-operate to regulate RNA metabolism. Several findings supported this hypothesis: (i) SMG6 and the piRNA-binding protein PIWIL1 have almost identical temporal expression and localization patterns, (ii) SMG6 and PIWIL1 physically interact, (iii) scores of genes upregulated in Smg6-cKO round spermatids overlap with those upregulated in Piwil1-KO round spermatids, and (iv) the phenotypic defects caused by SMG6 loss phenocopies the defects caused by PIWIL1 loss. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility. A germ cell-specific Smg6 conditional knockout mouse line (Smg6-cKO) was generated by crossing a mouse line harboring loxP sites on either side of Smg6 exon 9 with a transgenic mouse line expressing the Cre recombinase under the control of the Neurogenin 3 (Ngn3)-promoter. Wild type-like littermates were used as controls. RNA sequencing was performed to analyze differential expression of long poly(A)-containing RNAs and small RNAs in pachytene spermatocytes (Spc) and round spermatids (RS), as well as differential levels of long RNAs in chromatoid bodies (CB). 1) RNA-seq of CBs: CBs were isolated from Smg6-cKO and control testes using anti-DDX4 immunoaffinity protocol. Four control samples and three Smg6-cKO samples were analyzed. For each sample, material from 2-4 individual mice were pooled. 2) RNA-seq and 3) smallRNA-seq of testicular cells: Both testes from either one adult Smg6-cKO or one adult control mouse were used to isolate RS and Spc using BSA-gradient velocity sedimentation. For RNA-seq, three biological replicates were analyzed per genotype. For smallRNA-seq, three RS and five Spc biological replicates were analyzed per genotype.无义介导的RNA降解（Nonsense-mediated RNA decay, NMD）是一种保守的RNA周转通路。本研究报道了NMD通路中唯一的核酸内切酶SMG6对于雄性生殖系至关重要。生殖细胞条件性敲除（Germ-cell conditional knockout, cKO）Smg6会导致精子发生在单倍体早期阶段完全停滞。Smg6-cKO的圆形精子细胞会积累带有长3'非翻译区（3' untranslated regions, UTRs）的NMD靶mRNA，并且无法清除减数分裂（精子发生的前一阶段）中正常表达的转录本。SMG6与PIWI相互作用RNA（PIWI-interacting RNA, piRNA）通路组分在圆形小体（chromatoid body, CB）中高度富集，这是雄性生殖细胞中一种特化的细胞质颗粒。这引出了一个有趣的可能性：CB是SMG6与piRNA通路协同调控RNA代谢的位点。多项发现支持这一假说：(i) SMG6与piRNA结合蛋白PIWIL1具有几乎完全一致的时空表达和定位模式；(ii) SMG6与PIWIL1存在物理相互作用；(iii) Smg6-cKO圆形精子细胞中上调的大量基因与Piwil1-KO圆形精子细胞中的上调基因存在重叠；(iv) SMG6缺失导致的表型缺陷与PIWIL1缺失所引起的缺陷表型一致。综上，我们的研究结果表明SMG6是雄性生殖系转录组的关键调控因子，并阐明CB作为分子平台协调RNA调控通路以控制精子生成与生育能力。我们通过将带有Smg6外显子9两侧loxP位点的小鼠品系与在Neurogenin 3（Ngn3）启动子控制下表达Cre重组酶的转基因小鼠品系杂交，构建了生殖细胞特异性Smg6条件性敲除小鼠品系（Smg6-cKO）。以野生型同窝仔鼠作为对照。我们进行了RNA测序，以分析粗线期精母细胞（pachytene spermatocytes, Spc）和圆形精子细胞（round spermatids, RS）中长聚腺苷酸化RNA与小RNA的差异表达，以及圆形小体中长RNA的差异水平。1) 圆形小体RNA测序：通过抗DDX4免疫亲和分离方案从Smg6-cKO和对照小鼠的睾丸中分离圆形小体。共分析4份对照样本与3份Smg6-cKO样本，每份样本混合2-4只个体小鼠的材料。2) 睾丸细胞RNA测序与3) 小RNA测序：分别使用一只成年Smg6-cKO或成年对照小鼠的双侧睾丸，通过BSA梯度速度沉降法分离RS与Spc。对于RNA测序，每种基因型设置3个生物学重复。对于小RNA测序，每种基因型分别设置3份RS样本与5份Spc样本作为生物学重复。