The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.

Emerging roles and functional mechanisms of PIWI-interacting RNAs.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal germ line cells, piRNAs have essential roles in germ line development. The first function of PIWI-piRNA complexes to be described was the silencing of transposable elements, which is crucial for maintaining the integrity of the germ line genome. Later studies provided new insights into the functions of PIWI-piRNA complexes by demonstrating that they regulate protein-coding genes. Recent studies of piRNA biology, including in new model organisms such as golden hamsters, have deepened our understanding of both piRNA biogenesis and piRNA function. In this Review, we discuss the most recent advances in our understanding of piRNA biogenesis, the molecular mechanisms of piRNA function and the emerging roles of piRNAs in germ line development mainly in flies and mice, and in infertility, cancer and neurological diseases in humans.

Activating translation with phase separation.

Ribonucleoprotein granules allow activation of translation to complete mouse spermatogenesis.

RNF219 regulates CCR4-NOT function in mRNA translation and deadenylation.

Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

Functions of PIWI Proteins in Gene Regulation: New Arrows Added to the piRNA Quiver.

Piwi-interacting RNAs (piRNAs) and PIWI proteins play key functions in a wide range of biological and developmental processes through the regulation of cellular mRNAs, in addition to their role in transposable element (TE) repression. Evolutionary studies indicate that these PIWI functions in mRNA regulatory programs, occurring in both germ and somatic cells, are ancestral. Recent advances have widely expanded our understanding of these functions of PIWI proteins, identifying new mechanisms of action and strengthening their importance through their conservation in distant species. In this review, we discuss the latest findings regarding piRNA/PIWI-dependent mRNA decay in germ cells and during the maternal-to-zygotic transition in embryos combined with new modes of action of PIWI proteins in mRNA stabilization and translational activation and piRNA-independent roles of PIWI proteins in cancer.

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm.

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation.

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

piRNAs and PIWI proteins: regulators of gene expression in development and stem cells.

PIWI proteins and Piwi-interacting RNAs (piRNAs) have established and conserved roles in repressing transposable elements (TEs) in the germline of animals. However, in several biological contexts, a large proportion of piRNAs are not related to TE sequences and, accordingly, functions for piRNAs and PIWI proteins that are independent of TE regulation have been identified. This aspect of piRNA biology is expanding rapidly. Indeed, recent reports have revealed the role of piRNAs in the regulation of endogenous gene expression programs in germ cells, as well as in somatic tissues, challenging dogma in the piRNA field. In this Review, we focus on recent data addressing the biological and developmental functions of piRNAs, highlighting their roles in embryonic patterning, germ cell specification, stem cell biology, neuronal activity and metabolism.

Dicer-2 promotes mRNA activation through cytoplasmic polyadenylation.

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.

iCLIP of the PIWI Protein Aubergine in Drosophila Embryos.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs bound to specific Argonaute proteins, the PIWI proteins. piRNAs target mRNAs by complementarity to silence them; they play an important role in the repression of transposable elements in the germ line of many species. piRNAs and PIWI proteins are also involved in diverse biological processes through their role in the regulation of cellular mRNAs. In the Drosophila embryo, they contribute to the maternal mRNA decay occurring during the maternal-to-zygotic transition. CLIP (UV cross-linking and immunoprecipitation) techniques have been used to identify target mRNAs of Argonaute proteins. Here we describe the iCLIP (individual-nucleotide resolution CLIP) protocol that we have adapted for the PIWI protein Aubergine in Drosophila embryos.

piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm.

Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions.

Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl.

PIWI proteins play essential roles in germ cells and stem cell lineages. In Drosophila, Piwi is required in somatic niche cells and germline stem cells (GSCs) to support GSC self-renewal and differentiation. Whether and how other PIWI proteins are involved in GSC biology remains unknown. Here, we show that Aubergine (Aub), another PIWI protein, is intrinsically required in GSCs for their self-renewal and differentiation. Aub needs to be loaded with piRNAs to control GSC self-renewal and acts through direct mRNA regulation. We identify the Cbl proto-oncogene, a regulator of mammalian hematopoietic stem cells, as a novel GSC differentiation factor. Aub stimulates GSC self-renewal by repressing Cbl mRNA translation and does so in part through recruitment of the CCR4-NOT complex. This study reveals the role of piRNAs and PIWI proteins in controlling stem cell homeostasis via translational repression and highlights piRNAs as major post-transcriptional regulators in key developmental decisions.

Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression.

Measurement of mRNA Poly(A) Tail Lengths in Drosophila Female Germ Cells and Germ-Line Stem Cells.

mRNA regulation by poly(A) tail length variations plays an important role in many developmental processes. Recent advances have shown that, in particular, deadenylation (the shortening of mRNA poly(A) tails) is essential for germ-line stem cell biology in the Drosophila ovary. Therefore, a rapid and accurate method to analyze poly(A) tail lengths of specific mRNAs in this tissue is valuable. Several methods of poly(A) test (PAT) assays have been reported to measure mRNA poly(A) tail lengths in vivo. Here, we describe two of these methods (PAT and ePAT) that we have adapted for Drosophila ovarian germ cells and germ-line stem cells.

Translational Control of Autophagy by Orb in the Drosophila Germline.

Drosophila Orb, the homolog of vertebrate CPEB, is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb also has an essential function during early oogenesis that has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.

Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo.

The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Here, we perform iCLIP with the PIWI protein Aubergine (Aub) and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.

Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis.

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.