Processing of Alu small RNAs by DICER/ADAR1 complexes and their RNAi targets.

In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing-independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. Here, we report that ADAR1 together with DICER generates endogenous small RNAs, Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We identified AGO2-loaded Alu endo-siRNAs, which are highly expressed in commonly used cell lines. These Alu endo-siRNAs carrying both sense and antisense Alu sequences seem to target a set of genes containing a single Alu sequence, either antisense or sense, respectively, within their 3'UTR. In silico screening identified potential RNA silencing target genes for these Alu endo-siRNAs. We present results of a proof-of-concept experiment, in which sense Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3'UTRs and consequently induce apoptosis in HeLa cells. Our results clearly indicate that Alu endo-siRNAs are functional also in somatic cells.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis.

The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.

GPAT2 is required for piRNA biogenesis, transposon silencing, and maintenance of spermatogonia in mice†.

PIWI-interacting RNAs (piRNAs), a subclass of germ cell-specific noncoding small RNAs, are essential for de novo DNA methylation of retrotransposon genes in embryonic testes. PIWIL2/MILI, one of three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably via piRNA, and normal spermatogenesis. In vitro analysis using germline stem cells (GS cells) revealed that glycerol-3-phosphate acyltransferase 2 (GPAT2), which is a mitochondrial outer membrane protein involved in generation of lysophosphatidic acid (LPA) and highly expressed in testes, plays important roles in spermatogenesis. Namely, GPAT2 binds to PIWIL2 and is closely involved in the biogenesis of piRNAs; this process is independent of its enzymatic activity on LPA. However, GS cells recapitulate only a limited phase of spermatogenesis and the biological functions of GPAT2 remain largely unknown. In this study, we generated GPAT2-deficient mice and conducted comprehensive analyses. The deficient mice showed defective piRNA production and subsequent de-silencing of IAP and Line-1 retrotransposons in fetal testes. In addition, apoptosis of pachytene spermatocytes was observed. These abnormalities were all common to the phenotype of PIWIL2-deficient mice, in which piRNA production was impaired. GPAT2-deficient mice exhibited apoptosis in spermatogonia at the neonatal stage, which was not observed in PIWIL2-deficient mice. These data show that GPAT2 plays a critical role in preventing apoptosis in spermatogonia.

MVH in piRNA processing and gene silencing of retrotransposons.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals.

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25-40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (ℙ = 8 × 10(-3)-6 × 10(-8)). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Allogeneic offspring produced by induction of PD-L1 in spermatogonial stem cells via self-renewal stimulation.

The testis is an immune-privileged organ. It is considered that the testis somatic microenvironment is responsible for immune suppression. However, immunological properties of spermatogonial stem cells (SSCs) have remained unknown. Here, we report the birth of allogeneic offspring by enhanced expression of immunosuppressive PD-L1 in SSCs. In vitro supplementation of GDNF and FGF2 increased expression of PD-L1 in SSCs. Cultured SSCs maintained allogeneic spermatogenesis that persisted for >1 year. However, depletion or gene editing of Pd-l1 family genes in SSCs prevented allogeneic spermatogenesis, which suggested that germ cells are responsible for suppression of the allogeneic response. PD-L1 was induced by activation of the MAPK14-BCL6B pathway, which drives self-renewal by reactive oxygen species (ROS) generation. By contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1. Allogeneic offspring were born after SSC transplantation into congenitally infertile and chemically castrated mice. Thus, SSCs have unique immunological properties, which make allogeneic recipients into "surrogate fathers."

Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.

The role of RNA editing enzyme ADAR1 in human disease.

Adenosine deaminase acting on RNA (ADAR) catalyzes the posttranscriptional conversion of adenosine to inosine in double-stranded RNA (dsRNA), which can lead to the creation of missense mutations in coding sequences. Recent studies show that editing-dependent functions of ADAR1 protect dsRNA from dsRNA-sensing molecules and inhibit innate immunity and the interferon-mediated response. Deficiency in these ADAR1 functions underlie the pathogenesis of autoinflammatory diseases such as the type I interferonopathies Aicardi-Goutieres syndrome and dyschromatosis symmetrica hereditaria. ADAR1-mediated editing of endogenous coding and noncoding RNA as well as ADAR1 editing-independent interactions with DICER can also have oncogenic or tumor suppressive effects that affect tumor proliferation, invasion, and response to immunotherapy. The combination of proviral and antiviral roles played by ADAR1 in repressing the interferon response and editing viral RNAs alters viral morphogenesis and cell susceptibility to infection. This review analyzes the structure and function of ADAR1 with a focus on its position in human disease pathways and the mechanisms of its disease-associated effects. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.

Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 104 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility.

ADAR1 downregulation by autophagy drives senescence independently of RNA editing by enhancing p16INK4a levels.

Cellular senescence plays a causal role in ageing and, in mice, depletion of p16INK4a-expressing senescent cells delays ageing-associated disorders1,2. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms3,4. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16INK4a upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16INK4a-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16INK4a. Hence, downregulation of ADAR1 and SIRT1 mediates p16INK4a upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16INK4a expression.

ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells.

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3'UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5-MAVS-IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6-p38-MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3'-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3'-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.

Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA (dsRNA). Among the three types of mammalian ADARs, ADAR1 has long been recognized as an essential enzyme for normal development. The interferon-inducible ADAR1p150 is involved in immune responses to both exogenous and endogenous triggers, whereas the functions of the constitutively expressed ADAR1p110 are variable. Recent findings that ADAR1 is involved in the recognition of self versus non-self dsRNA provide potential explanations for its links to hematopoiesis, type I interferonopathies, and viral infections. Editing in both coding and noncoding sequences results in diseases ranging from cancers to neurological abnormalities. Furthermore, editing of noncoding sequences, like microRNAs, can regulate protein expression, while editing of Alu sequences can affect translational efficiency and editing of proximal sequences. Novel identifications of long noncoding RNA and retrotransposons as editing targets further expand the effects of A-to-I editing. Besides editing, ADAR1 also interacts with other dsRNA-binding proteins in editing-independent manners. Elucidating the disease-specific patterns of editing and/or ADAR1 expression may be useful in making diagnoses and prognoses. In this review, we relate the mechanisms of ADAR1's actions to its pathological implications, and suggest possible mechanisms for the unexplained associations between ADAR1 and human diseases.

Comprehensive DNA Methylation Analysis of Retrotransposons in Male Germ Cells.

De novo DNA methylation of retrotransposons is critical for silencing. Here, we use DNA methylation analysis to examine retrotransposons in mouse male germ cells. DNA methylation of long interspersed nuclear elements (LINEs) is dependent on piRNA, and younger LINEs exhibit greater piRNA dependence. In contrast, most long terminal repeat (LTR) retrotransposons produce lower levels of piRNAs and do not show significant piRNA dependence. The relationship between DNA methylation and corresponding piRNA expression of several LTR retrotransposons was reduced in Mili-null cells, but not Miwi2-null cells. These observations raise the possibility of piRNA-dependent DNA methylation without Miwi2. Therefore, it appears that the molecular mechanisms of the gene silencing of retrotransposons are more complicated than previously thought.

Induction of DNA methylation by artificial piRNA production in male germ cells.

Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.

DNA methylation in mouse testes.

DNA methylation of retrotransposons and imprinted genes is accurately regulated in spermatogenesis. In particular, CpG methylation of long interspersed elements-1 (LINE1 or L1) and intracisternal A-particle (IAP) retrotransposons during spermatogenesis has been well characterized. CpG methylation of the regulatory regions of retrotransposons is acquired during embryonic testis development; however, reductions of DNA methylation in LINE1 and/or IAP and/or Rasgrf1, which is an imprinted gene, are observed in deficient mice of piRNA biogenesis concerning. Here, we describe two methods, bisulfite sequencing and Southern blotting using a methylation-sensitive restriction enzyme, for analysis of DNA methylation of LINE1, IAP, and imprinted genes in mouse testes.