Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs. piRNA sequencing, RNA immunoprecipitation, and expression measurements (RNA-Seq and ribosome profiling) in wild-type and Mael -/- testes
Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs.
Project description:PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. Previously, we showed that ribosomes guide the biogenesis of piRNAs from long non-coding RNAs (lncRNAs) after translating the short open reading frames (ORFs) near their 5′ cap. It remained unclear, however, how ribosomes proceed downstream of ORFs and how piRNA precursors distinguish from other RNAs. It is thus important to test whether a short ORF length is required for substrate recognition for ribosome guided-piRNA biogenesis. Here, we characterized a poorly understood class of piRNAs that originate from the 3′ untranslated regions (3′UTRs) of protein coding genes in mice and chickens. We demonstrate that their precursors are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3′UTRs after translation of upstream long ORFs. Similar to non-sense mediated decay (NMD), piRNA biogenesis degrades mRNA right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Interestingly, however, we found that NMD, along with other surveillance pathways for ribosome recycling are temporally sequestered during the pachytene stage to allow for robust piRNA production. Although 3′UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded transposable elements (TEs) and produce piRNAs that cleave TEs, suggesting that TE suppression, rather than the function of proteins, is the primary evolutionary force maintaining a subset of mRNAs as piRNA precursors. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved, general piRNA biogenesis mechanism that recognizes translating RNAs regardless of their ORF length in amniotes.
Project description:PIWI-interacting RNAs (piRNAs) are small non-coding RNAs essential for animal germ cell development. Despite intense investigation of post-transcriptional processing, piRNA biogenesis at the chromatin level remains enigmatic. Here we document that BTBD18 is a pachytene nuclear protein in mouse testes that occupies intergenic pachytene piRNA-producing loci with remarkable genome-wide specificity. Deficiency of Btbd18 in mice results in disrupted piRNA biogenesis and male sterility. Transcriptome profiling, chromatin accessibility and RNA polymerase II occupancy demonstrate that BTBD18 facilitates expression of pachytene piRNA precursors by promoting transcription elongation. Therefore, we conclude that BTBD18 functionally licenses genomic sources of piRNAs for transcription activation in mice, and propose that piRNA precursor biogenesis is regulated by transcriptional elongation.
Project description:In the fetal mouse testis, PIWI Interacting RNAs (piRNAs) guide PIWI proteins to silence transposons, but after birth, most post-pubertal pachytene piRNAs map to genome uniquely and are thought to regulate genes required for male fertility. In human males, the developmental classes, precise genomic origins, and transcriptional regulation of post-natal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce post-natal piRNAs in human juvenile and adult testes. As in mouse, A‑MYB in humans drives transcription of both pachytene piRNA precursor transcripts and the mRNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that during mammalian evolution, pachytene piRNA genes are under fewer selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. Transcriptome and ChIP sequencing in mouse and rooster testes
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PAS-Seq and CAGE in mouse testes
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. ChIP sequencing in mouse and rooster testes.
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. smallRNA-Seq in mouse and rooster testes