The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex.

In the neocortex, functionally distinct areas process specific types of information. Area identity is established by morphogens and transcriptional master regulators, but downstream mechanisms driving area-specific neuronal specification remain unclear. Here, we reveal a role for RNA-binding proteins in defining area-specific cytoarchitecture. Mice lacking Pum2 or overexpressing human TDP-43 show apparent 'motorization' of layers IV and V of primary somatosensory cortex (S1), characterized by dramatic expansion of cells co-expressing Sox5 and Bcl11b/Ctip2, a hallmark of subcerebral projection neurons, at the expense of cells expressing the layer IV neuronal marker Rorß. Moreover, retrograde labeling experiments with cholera toxin B in Pum2; Emx1-Cre and TDP43A315T mice revealed a corresponding increase in subcerebral connectivity of these neurons in S1. Intriguingly, other key features of somatosensory area identity are largely preserved, suggesting that Pum2 and TDP-43 may function in a downstream program, rather than controlling area identity per se. Transfection of primary neurons and in utero electroporation (IUE) suggest cell-autonomous and post-mitotic modulation of Sox5, Bcl11b/Ctip2, and Rorß levels. Mechanistically, we find that Pum2 and TDP-43 directly interact with and affect the translation of mRNAs encoding Sox5, Bcl11b/Ctip2, and Rorß. In contrast, effects on the levels of these mRNAs were not detectable in qRT-PCR or single-molecule fluorescent in situ hybridization assays, and we also did not detect effects on their splicing or polyadenylation patterns. Our results support the notion that post-transcriptional regulatory programs involving translational regulation and mediated by Pum2 and TDP-43 contribute to elaboration of area-specific neuronal identity and connectivity in the neocortex.

SYNGR4 and PLEKHB1 deregulation in motor neurons of amyotrophic lateral sclerosis models: potential contributions to pathobiology.

Amyotrophic lateral sclerosis is the most common adult-onset neurodegenerative disease affecting motor neurons. Its defining feature is progressive loss of motor neuron function in the cortex, brainstem, and spinal cord, leading to paralysis and death. Despite major advances in identifying genes that can cause disease when mutated and model the disease in animals and cellular models, it still remains unclear why motor symptoms suddenly appear after a long pre-symptomatic phase of apparently normal function. One hypothesis is that age-related deregulation of specific proteins within key cell types, especially motor neurons themselves, initiates disease symptom appearance and may also drive progressive degeneration. Genome-wide in vivo cell-type-specific screening tools are enabling identification of candidates for such proteins. In this minireview, we first briefly discuss the methodology used in a recent study that applied a motor neuron-specific RNA-Seq screening approach to a standard model of TAR DNA-binding protein-43 (TDP-43)-driven amyotrophic lateral sclerosis. A key finding of this study is that synaptogyrin-4 and pleckstrin homology domain-containing family B member 1 are also deregulated at the protein level within motor neurons of two unrelated mouse models of mutant TDP-43 driven amyotrophic lateral sclerosis. Guided by what is known about molecular and cellular functions of these proteins and their orthologs, we outline here specific hypotheses for how changes in their levels might potentially alter cellular physiology of motor neurons and detrimentally affect motor neuron function. Where possible, we also discuss how this information could potentially be used in a translational context to develop new therapeutic strategies for this currently incurable, devastating disease.

Corrigendum: Emerging Evidence of Translational Control by AU-Rich Element-Binding Proteins.

[This corrects the article DOI: 10.3389/fgene.2019.00332.].

Motor neuron translatome reveals deregulation of SYNGR4 and PLEKHB1 in mutant TDP-43 amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease with progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS, and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined translating ribosome affinity purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43-driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wild type hTDP-43, we identified hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated the deregulated candidates Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs, we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they are general features of TDP-43-driven ALS. Thus, we identified SYNGR4 and PLEKHB1 to be deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that these proteins could be functionally important for driving the transition to the symptomatic phase of the disease.

ARE-binding protein ZFP36L1 interacts with CNOT1 to directly repress translation via a deadenylation-independent mechanism.

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.

Emerging Evidence of Translational Control by AU-Rich Element-Binding Proteins.

RNA-binding proteins (RBPs) are key regulators of posttranscriptional gene expression and control many important biological processes including cell proliferation, development, and differentiation. RBPs bind specific motifs in their target mRNAs and regulate mRNA fate at many steps. The AU-rich element (ARE) is one of the major cis-regulatory elements in the 3' untranslated region (UTR) of labile mRNAs. Many of these encode factors requiring very tight regulation, such as inflammatory cytokines and growth factors. Disruption in the control of these factors' expression can cause autoimmune diseases, developmental disorders, or cancers. Therefore, these mRNAs are strictly regulated by various RBPs, particularly ARE-binding proteins (ARE-BPs). To regulate mRNA metabolism, ARE-BPs bind target mRNAs and affect some factors on mRNAs directly, or recruit effectors, such as mRNA decay machinery and protein kinases to target mRNAs. Importantly, some ARE-BPs have stabilizing roles, whereas others are destabilizing, and ARE-BPs appear to compete with each other when binding to target mRNAs. The function of specific ARE-BPs is modulated by posttranslational modifications (PTMs) including methylation and phosphorylation, thereby providing a means for cellular signaling pathways to regulate stability of specific target mRNAs. In this review, we summarize recent studies which have revealed detailed molecular mechanisms of ARE-BP-mediated regulation of gene expression and also report on the importance of ARE-BP function in specific physiological contexts and how this relates to disease. We also propose an mRNP regulatory network based on competition between stabilizing ARE-BPs and destabilizing ARE-BPs.

Bassoon proteinopathy drives neurodegeneration in multiple sclerosis.

Multiple sclerosis (MS) is characterized by inflammatory insults that drive neuroaxonal injury. However, knowledge about neuron-intrinsic responses to inflammation is limited. By leveraging neuron-specific messenger RNA profiling, we found that neuroinflammation leads to induction and toxic accumulation of the synaptic protein bassoon (Bsn) in the neuronal somata of mice and patients with MS. Neuronal overexpression of Bsn in flies resulted in reduction of lifespan, while genetic disruption of Bsn protected mice from inflammation-induced neuroaxonal injury. Notably, pharmacological proteasome activation boosted the clearance of accumulated Bsn and enhanced neuronal survival. Our study demonstrates that neuroinflammation initiates toxic protein accumulation in neuronal somata and advocates proteasome activation as a potential remedy.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease.

The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor.

Many viruses strongly prefer to infect certain cell types, a phenomenon known as "tropism." Understanding tropism's molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5' untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3' end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder.

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.

De Novo Mutations in DENR Disrupt Neuronal Development and Link Congenital Neurological Disorders to Faulty mRNA Translation Re-initiation.

Disruptions to neuronal mRNA translation are hypothesized to underlie human neurodevelopmental syndromes. Notably, the mRNA translation re-initiation factor DENR is a regulator of eukaryotic translation and cell growth, but its mammalian functions are unknown. Here, we report that Denr influences the migration of murine cerebral cortical neurons in vivo with its binding partner Mcts1, whereas perturbations to Denr impair the long-term positioning, dendritic arborization, and dendritic spine characteristics of postnatal projection neurons. We characterized de novo missense mutations in DENR (p.C37Y and p.P121L) detected in two unrelated human subjects diagnosed with brain developmental disorder to find that each variant impairs the function of DENR in mRNA translation re-initiation and disrupts the migration and terminal branching of cortical neurons in different ways. Thus, our findings link human brain disorders to impaired mRNA translation re-initiation through perturbations in DENR (OMIM: 604550) function in neurons.

The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons.

Dendrites exhibit enormous diversity in form and can differ in size by several orders of magnitude even in a single animal. However, whether neurons with large dendrite arbors have specialized mechanisms to support their growth demands is unknown. To address this question, we conducted a genetic screen for mutations that differentially affected growth in neurons with different-sized dendrite arbors. From this screen, we identified a mutant that selectively affects dendrite growth in neurons with large dendrite arbors without affecting dendrite growth in neurons with small dendrite arbors or the animal overall. This mutant disrupts a putative amino acid transporter, Pathetic (Path), that localizes to the cell surface and endolysosomal compartments in neurons. Although Path is broadly expressed in neurons and nonneuronal cells, mutation of path impinges on nutrient responses and protein homeostasis specifically in neurons with large dendrite arbors but not in other cells. Altogether, our results demonstrate that specialized molecular mechanisms exist to support growth demands in neurons with large dendrite arbors and define Path as a founding member of this growth program.

DENR-MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth.

During cap-dependent eukaryotic translation initiation, ribosomes scan messenger RNA from the 5' end to the first AUG start codon with favourable sequence context. For many mRNAs this AUG belongs to a short upstream open reading frame (uORF), and translation of the main downstream ORF requires re-initiation, an incompletely understood process. Re-initiation is thought to involve the same factors as standard initiation. It is unknown whether any factors specifically affect translation re-initiation without affecting standard cap-dependent translation. Here we uncover the non-canonical initiation factors density regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT-1; also called MCTS1 in humans) as the first selective regulators of eukaryotic re-initiation. mRNAs containing upstream ORFs with strong Kozak sequences selectively require DENR-MCT-1 for their proper translation, yielding a novel class of mRNAs that can be co-regulated and that is enriched for regulatory proteins such as oncogenic kinases. Collectively, our data reveal that cells have a previously unappreciated translational control system with a key role in supporting proliferation and tissue growth.

A novel mouse model for inhibition of DOHH-mediated hypusine modification reveals a crucial function in embryonic development, proliferation and oncogenic transformation.

The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh-/- cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.

Automated high-throughput RNAi screening in human cells combined with reporter mRNA transfection to identify novel regulators of translation.

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.

Protein-protein-interaction network organization of the hypusine modification system.

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.

The SXL-UNR corepressor complex uses a PABP-mediated mechanism to inhibit ribosome recruitment to msl-2 mRNA.

Drosophila female viability requires translational repression of msl-2 mRNA by the SXL-UNR 3' UTR corepressor complex, which inhibits ribosome recruitment by an unknown mechanism. Here, we reveal a key role for the poly(A)-binding protein (PABP), a translational activator, in this inhibitory mechanism. Efficient msl-2 mRNA silencing via the 3' UTR requires both a poly(A) tail and PABP function, and we find that UNR directly interacts with PABP. To investigate how the repressor complex and PABP affect RNP composition during early steps in translation initiation, we established direct biochemical assays for synergistic recruitment of eIF4F and ribosomes by the cap and poly(A) tail. We find that the repressor complex targets ribosome binding after PABP-mediated recruitment of eIF4E/G. Our results uncover an important regulatory mechanism of Drosophila dosage compensation and provide insight into PABP-dependent translational control by 3' UTR-bound regulatory proteins.

Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression.

Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. Here we use a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, we identified defined modifications of the triphosphate backbone that augment miRNA-mediated inhibition of translation initiation but are "neutral" toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. Our data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators.