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In animals, PIWI-interacting RNAs (piRNAs) of 21–35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.

The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation. The PIWI protein MIWI2 counteracts transposon activity by transcriptional silencing in the mammalian germline. Here, the authors show that TEX15 interacts with MIWI2 and is required for piRNA-directed methylation of transposable elements in male germ cells.

MicroRNAs (miRNAs) are small, noncoding RNAs that mediate posttranscriptional gene suppression in a sequence-specific manner. The ability of a single miRNA species to target multiple messenger RNAs (mRNAs) makes miRNAs exceptionally important regulators of various cellular functions. The regulatory capacity of miRNAs is increased further by the miRNA ability to suppress gene expression using multiple mechanisms that range from translational inhibition to mRNA degradation. The high miRNA diversity multiplied by the large number of individual miRNA targets generates a vast regulatory RNA network than enables flexible control of mRNA expression. The gene-regulatory capacity and diversity of miRNAs is particularly valuable in the brain, where functional specialization of neurons and persistent flow of information requires constant neuronal adaptation to environmental cues. In this review we will summarize the current knowledge about miRNA biogenesis and miRNA expression regulation with a focus on the role of miRNAs in the mammalian nervous system.

microRNAs (miRNAs) repress target transcripts through partial complementarity. By contrast, highly complementary miRNA-binding sites within viral and artificially engineered transcripts induce miRNA degradation in vitro and in cell lines. Here, we show that a genome-encoded transcript harboring a near-perfect and deeply conserved miRNA-binding site for miR-29 controls zebrafish and mouse behavior. This transcript originated in basal vertebrates as a long noncoding RNA (lncRNA) and evolved to the protein-coding gene NREP in mammals, where the miR-29-binding site is located within the 3′ UTR. We show that the near-perfect miRNA site selectively triggers miR-29b destabilization through 3′ trimming and restricts its spatial expression in the cerebellum. Genetic disruption of the miR-29 site within mouse Nrep results in ectopic expression of cerebellar miR-29b and impaired coordination and motor learning. Thus, we demonstrate an endogenous target-RNA-directed miRNA degradation event and its requirement for animal behavior.

TUT4 and TUT7 mediate 3′ uridylation of mRNA transcripts, preferentially those with short poly(A) tails; in the absence of TUT4 and TUT7, oocytes cannot mature and female mice are infertile. Maternal transcriptome makers After fertilization, early embryonic events are dictated by the maternal messenger RNAs stored in the oocyte. Dónal O'Carroll and colleagues show that the appropriate maternal transcriptome is achieved via an essential uridylation process that eliminates unwanted transcripts. The terminal 3′ uridylation is mediated by TUT4/7 complex, and this modification ensures the correct polyadenylation (poly(A)) tail length. In the absence of TUT4/7, oocytes cannot mature, and fertilization is prevented. This study reveals the specific and essential function of poly(A) tail length in sculpting and activating the functional maternal transcriptome. A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome 1 , 2 . How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3′ terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3′ oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3′ terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation 1 . In the male germline, RNA-directed DNA methylation silences young, active transposable elements 2 – 4 . The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements 3 , 5 . piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation. Newly identified protein SPOCD1 is crucial in de novo DNA methylation directed by PIWI proteins and piRNAs, helping to control DNA silencing in mouse male germline.

The control of motor behavior in animals and humans requires constant adaptation of neuronal networks to signals of various types and strengths. We found that microRNA-128 (miR-128), which is expressed in adult neurons, regulates motor behavior by modulating neuronal signaling networks and excitability. miR-128 governs motor activity by suppressing the expression of various ion channels and signaling components of the extracellular signal-regulated kinase ERK2 network that regulate neuronal excitability. In mice, a reduction of miR-128 expression in postnatal neurons causes increased motor activity and fatal epilepsy. Overexpression of miR-128 attenuates neuronal responsiveness, suppresses motor activity, and alleviates motor abnormalities associated with Parkinson's-like disease and seizures in mice. These data suggest a therapeutic potential for miR-128 in the treatment of epilepsy and movement disorders.

Several developmental stages of spermatogenesis are transcriptionally quiescent which presents major challenges associated with the regulation of gene expression. Here we identify that the zygotene to pachytene transition is not only associated with the resumption of transcription but also a wave of programmed mRNA degradation that is essential for meiotic progression. We explored whether terminal uridydyl transferase 4- (TUT4-) or TUT7-mediated 3′ mRNA uridylation contributes to this wave of mRNA degradation during pachynema. Indeed, both TUT4 and TUT7 are expressed throughout most of spermatogenesis, however, loss of either TUT4 or TUT7 does not have any major impact upon spermatogenesis. Combined TUT4 and TUT7 (TUT4/7) deficiency results in embryonic growth defects, while conditional gene targeting revealed an essential role for TUT4/7 in pachytene progression. Loss of TUT4/7 results in the reduction of miRNA, piRNA and mRNA 3′ uridylation. Although this reduction does not greatly alter miRNA or piRNA expression, TUT4/7-mediated uridylation is required for the clearance of many zygotene-expressed transcripts in pachytene cells. We find that TUT4/7-regulated transcripts in pachytene spermatocytes are characterized by having long 3′ UTRs with length-adjusted enrichment for AU-rich elements. We also observed these features in TUT4/7-regulated maternal transcripts whose dosage was recently shown to be essential for sculpting a functional maternal transcriptome and meiosis. Therefore, mRNA 3′ uridylation is a critical determinant of both male and female germline transcriptomes. In conclusion, we have identified a novel requirement for 3′ uridylation-programmed zygotene mRNA clearance in pachytene spermatocytes that is essential for male meiotic progression.

Transposon silencing by Piwi proteins The combination of Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) mediates epigenetic transposon silencing in animal germlines. Piwi proteins are predicted to be endonucleases, but the significance of this activity had not been demonstrated in vivo . The laboratories of Dónal O'Carroll and Ramesh Pillai have now made mouse models in which residues expected to be critical for nuclease activity in the three mouse Piwi homologues, Mili, Miwi and Miwi2, are mutated. The mutant mice show phenotypic differences. The Mili and Miwi mutants are defective in piRNA production, transposon silencing and fertility, whereas the Miwi2 mutant has normal piRNA levels, seems to undergo piRNA amplification and silences transposons. These studies highlight distinctions between the murine enzymes responsible for piRNA biogenesis. Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing 1 . The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line 2 , 3 , 4 . Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis 5 , 6 , 7 ; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili DAH and Miwi2 DAH alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili DAH fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili DAH mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2 DAH mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2−/− and Dnmt3l−/− spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2−/− and Dnmt3l−/− mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction.