Almost half of the human genome consists of retrotransposons—‘parasitic’ sequences that insert themselves into the host genome via an RNA intermediate. Although most of these sequences are silenced or mutationally deactivated, they can present opportunities for evolutionary innovation: mutation of a deteriorating retrotransposon can result in a gene that provides a selective advantage to the host in a process termed ‘domestication’1–3. The PNMA family of gag-like capsid genes was domesticated from an ancient vertebrate retrotransposon of the Metaviridae clade at least 100 million years ago4,5. PNMA1 and PNMA4 are positively regulated by the master germ cell transcription factors MYBL1 and STRA8, and their transcripts are bound by the translational regulator DAZL during gametogenesis6. This developmental regulation of PNMA1 and PNMA4 expression in gonadal tissue suggested to us that they might serve a reproductive function. Through the analysis of donated human ovaries, genome-wide association studies (GWASs) and mouse models, we found that PNMA1 and PNMA4 are necessary for the maintenance of a normal reproductive lifespan. These proteins self-assemble into capsid-like structures that exit human cells, and we observed large PNMA4 particles in mouse male gonadal tissue that contain RNA and are consistent with capsid formation. PNMA-family genes are gag-like elements of ancient retrotransposon origin whose roles in mammalian genomes is incompletely understood. Wood et al. found that two retrotransposon-derived proteins, PNMA1 and PNMA4, support mammalian fertility in both sexes and become increasingly important during aging. These proteins assemble into capsid-like structures that contain RNA and exit human cells.

Different mutations in the RNA‐binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late‐onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA‐binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1‐specific targets, but the severe mutation disrupts interactions with several RNA‐binding proteins and the regulation of their targets. In patient‐derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA‐binding proteins in a physiological context will require studying their interactions as well as their targets.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3′ untranslated region (3′UTR), introns, or exons. Most studies focus on APA within the 3′UTR; however, here, we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3′UTR and internal sites in a pathway-specific manner. Genes associated with neuronal function undergo mostly intronic APA, reducing their expression, while genes associated with heart and skeletal function mostly undergo 3′UTR APA and are up-regulated. This suggests that, under healthy conditions, cells toggle between internal and 3′UTR APA to modulate protein expression.

Mutations in the RNA-binding protein (RBP) Pumilio1 (PUM1) can cause dramatically different phenotypes. We previously noted that phenotypic severity tracked with protein dosage: a mild mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas PUM1 haploinsufficiency causes developmental delay and seizures. Why this difference in expression should cause such different phenotypes has been unclear: PUM1 targets are de-repressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1’s RNA-binding ability. We therefore developed a PUM1 interactome in the murine brain. We find that mild PUM1 loss de-represses PUM1-specific targets, but PUM1 haploinsufficiency disrupts several interactors and regulation of their targets. We validated these phenomena in patient-derived cell lines and show that normalizing PUM1 levels restores interactors and their targets to proper levels. We therefore propose that dosage sensitivity does not necessarily reflect a linear change in protein abundance but can involve distinct mechanisms. Studying the interactors of RBPs in vivo will be necessary to understand their functions in neurological diseases.

Publishing a primary research article is typically the result of a collaborative effort between a variety of researchers across differing career stages. STAR Protocols can complement a research article and empower authors to share the expertise they contributed to the larger study. In this Backstory, we interview members of the Gennarino lab, who published a Cell paper and four protocols, covering bioinformatics, culturing of patient-derived cell lines, neuroimaging from mouse brain sections and primary neurons, and mouse seizure recordings.For more information on the protocols related to this backstory, please refer to (Gennarino et al., 2018).

Despite being among the largest neurons in the mammalian brain, Purkinje cells are difficult to visualize and trace via immunofluorescence because their dendritic arbors extend through several cerebellar layers. This protocol describes a two-antibody strategy we developed to study Purkinje cell morphology in mice. With it, one can reconstruct three-dimensional images of Purkinje cells at single-neuron resolution across multiple layers. The substantially improved image quality reveals subtle defects, enabling more meaningful morphological analysis. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2015).

Quantifying differences in the amount of protein and mRNA caused by missense mutations in a gene of interest can be challenging, especially when using patient-derived primary cells, which are intrinsically variable. In this protocol, we describe how to culture patient-derived lymphoblast and fibroblast cell lines for later mRNA and protein quantification. We also describe the steps to examine variants of PUM1 in HEK293T cells, but the protocol can be applied to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Background MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. Methods "Bottom-of-the-crypt" (EPCAM(+)/CD44(+)/CD66a(low)) and "top-of-the-crypt" (EPCAM(+)/CD44(neg)/CD66a(high)) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derivedxenografts (PDX). Results Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. Conclusion In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

Genetic variants that affect neurological function will often produce changes visible at the level of gross morphology, either of the whole brain or of specific neuronal types. Here we describe how to perfuse and dissect the brain in preparation for Nissl staining. Then we outline steps for culturing mouse primary hippocampal neurons to evaluate dendritic arborization ( Sholl analysis). For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Recurrent seizures are a common feature in many neurologic disorders. Seizure examination may help with diagnosis, preclinical study, and development of treatment strategies. Here we detail protocols to prepare and implant electrodes, as well as to record and analyze seizure events in freely moving mice. SCA47 mice exhibit both preclinical seizures (i.e., epileptiform discharges of EEG) starting from ∼14 weeks of age and behavioral seizures (i.e., spontaneous behavioral seizures) starting from ∼22 weeks of age. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Identifying a disease gene and determining its causality in patients can be challenging. Here, we present an approach to predicting the pathogenicity of deletions and missense variants for an autosomal dominant gene. We provide online resources for identifying patients and determining constraint metrics to isolate the causal gene among several candidates encompassed in a shared region of deletion. We also provide instructions for optimizing functional annotation programs that may be otherwise inaccessible to a nonexpert or novice in computational approaches. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Mutations in the RNA-binding protein (RBP) Pumilio1 (PUM1) can cause dramatically different phenotypes. Mild mutations that reduce PUM1 levels by 25% lead to a mild, adult-onset ataxia, whereas more severe mutations that reduce PUM1 levels 40-60% produce an infantile syndrome involving multiple developmental delays and seizures. Why this difference in expression should cause such different phenotypes has been unclear; known PUM1 targets are de-repressed to equal degrees in both diseases. We therefore sought to identify PUM1’s protein partners in the murine brain. We identified a number of putative interactors involved in different aspects of RNA metabolism such as silencing, alternative splicing, and polyadenylation. We find that PUM1 haploinsufficiency alters the stability of several interactors and disrupts the regulation of targets of those interactors, whereas mild PUM1 loss only de-represses PUM1-specific targets. We validated these phenomena in patient-derived cell lines and show that normalizing PUM1 levels rescues the levels of interactors and their targets. We therefore propose that dosage sensitivity does not necessarily reflect a linear change in levels but can involve distinct mechanisms. Studying the interactors of RBPs in vivo will be necessary to understand their functions in neurological diseases.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Identifying modifiers of dosage-sensitive genes involved in neurodegenerative disorders is imperative to discover novel genetic risk factors and potential therapeutic entry points. In this study, we focus on Ataxin-1 (ATXN1), a dosage-sensitive gene involved in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1). While the precise maintenance of ATXN1 levels is essential to prevent disease, the mechanisms that regulate ATXN1 ex pression remain largely unknown. We demonstrate that ATXN1's unusually long 5 ' untranslated region (5 ' UTR) negatively regulates its expression via posttranscriptional mechanisms. Based on recent reports that microRNAs (miRNAs) can interact with both 3 ' and 5 ' UTRs to regulate their target genes, we identify miR760 as a negative regulator that binds to a conserved site in ATXN1's 5 ' UTR to induce RNA degradation and translational inhibition. We found that delivery of Adeno-associated virus (AAV)-expressing miR760 in the cerebellum reduces ATXN1 levels in vivo and mitigates motor coordination deficits in a mouse model of SCA1. These findings provide new insights into the regulation of ATXN1 levels, present additional evidence for miRNA-mediated gene regulation via 5 ' UTR binding, and raise the possibility that noncoding mutations in the ATXN1 locus may act as risk factors for yet to be discovered progressive ataxias.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aβ deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aβ pathology, rendering it a potential contributor to AD risk and pathogenesis.

RNA splicing entails the coordinated interaction of more than 150 proteins in the spliceosome, one of the most complex of the cell’s molecular machines. We previously discovered that the RNA-binding motif protein 17 (RBM17), a component of the spliceosome, is essential for survival and cell maintenance. Here, we find that it interacts with the spliceosomal factors U2SURP and CHERP and that they reciprocally regulate each other’s stability, both in mouse and in human cells. Individual knockdown of each of the three proteins induces overlapping changes in splicing and gene expression of transcripts enriched for RNA-processing factors. Our results elucidate the function of RBM17, U2SURP, and CHERP and link the activity of the spliceosome to the regulation of downstream RNA-binding proteins. These data support the hypothesis that, beyond driving constitutive splicing, spliceosomal factors can regulate alternative splicing of specific targets.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.