The SYNGAP1 3'UTR Variant in ALS Patients Causes Aberrant SYNGAP1 Splicing and Dendritic Spine Loss by Recruiting HNRNPK.

Fused in sarcoma (FUS) is a pathogenic RNA-binding protein in amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilizes Synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains spine maturation. To elucidate the pathologic roles of this mechanism in ALS patients, we identified the SYNGAP1 3'UTR variant rs149438267 in seven (four males and three females) out of 807 ALS patients at the FUS binding site from a multicenter cohort in Japan. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, increased isoform α1 levels, and decreased isoform γ levels, which caused dendritic spine loss. Moreover, the SYNGAP1 variant excessively recruited FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK), and antisense oligonucleotides (ASOs) blocking HNRNPK altered aberrant splicing and ameliorated dendritic spine loss. These data suggest that excessive recruitment of RNA-binding proteins, especially HNRNPK, as well as changes in SYNGAP1 isoforms, are crucial for spine formation in motor neurons.SIGNIFICANCE STATEMENT It is not yet known which RNAs cause the pathogenesis of amyotrophic lateral sclerosis (ALS). We previously reported that Fused in sarcoma (FUS), a pathogenic RNA-binding protein in ALS, stabilizes synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains dendritic spine maturation. To elucidate whether this mechanism is crucial for ALS, we identified the SYNGAP1 3'UTR variant rs149438267 at the FUS binding site. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, which caused dendritic spine loss along with excessive recruitment of FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK). Our findings that dendritic spine loss is because of excess recruitment of RNA-binding proteins provide a basis for the future exploration of ALS-related RNA-binding proteins.

Failure to Degrade CAT-Tailed Proteins Disrupts Neuronal Morphogenesis and Cell Survival.

Ribosome-associated quality control (RQC) relieves stalled ribosomes and eliminates potentially toxic nascent polypeptide chains (NCs) that can cause neurodegeneration. During RQC, RQC2 modifies NCs with a C-terminal alanine and threonine (CAT) tail. CAT tailing promotes ubiquitination of NCs for proteasomal degradation, while RQC failure in budding yeast disrupts proteostasis via CAT-tailed NC aggregation. However, the CAT tail and its cytotoxicity in mammals have remained largely uncharacterized. We demonstrate that NEMF, a mammalian RQC2 homolog, modifies translation products of nonstop mRNAs, major erroneous mRNAs in mammals, with a C-terminal tail mainly composed of alanine with several other amino acids. Overproduction of nonstop mRNAs induces NC aggregation and caspase-3-dependent apoptosis and impairs neuronal morphogenesis, which are ameliorated by NEMF depletion. Moreover, we found that homopolymeric alanine tailing at least partially accounts for CAT-tail cytotoxicity. These findings explain the cytotoxicity of CAT-tailed NCs and demonstrate physiological significance of RQC on proper neuronal morphogenesis and cell survival.

TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1.

Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.

3'UTR Length-Dependent Control of SynGAP Isoform α2 mRNA by FUS and ELAV-like Proteins Promotes Dendritic Spine Maturation and Cognitive Function.

FUS is an RNA-binding protein associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated intrinsic roles of FUS in synaptic function. However, the mechanism underlying FUS's regulation of synaptic morphology has remained unclear. We found that reduced mature spines after FUS depletion were associated with the internalization of PSD-95 within the dendritic shaft. Mass spectrometry of PSD-95-interacting proteins identified SynGAP, whose expression decreased after FUS depletion. Moreover, FUS and the ELAV-like proteins ELAVL4 and ELAVL1 control SynGAP mRNA stability in a 3'UTR length-dependent manner, resulting in the stable expression of the alternatively spliced SynGAP isoform α2. Finally, abnormal spine maturation and FTLD-like behavioral deficits in FUS-knockout mice were ameliorated by SynGAP α2. Our findings establish an important link between FUS and ELAVL proteins for mRNA stability control and indicate that this mechanism is crucial for the maintenance of synaptic morphology and cognitive function.

Ubiquitination of stalled ribosome triggers ribosome-associated quality control.

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.

Altered Tau Isoform Ratio Caused by Loss of FUS and SFPQ Function Leads to FTLD-like Phenotypes.

Fused in sarcoma (FUS) and splicing factor, proline- and glutamine-rich (SFPQ) are RNA binding proteins that regulate RNA metabolism. We found that alternative splicing of the Mapt gene at exon 10, which generates 4-repeat tau (4R-T) and 3-repeat tau (3R-T), is regulated by interactions between FUS and SFPQ in the nuclei of neurons. Hippocampus-specific FUS- or SFPQ-knockdown mice exhibit frontotemporal lobar degeneration (FTLD)-like behaviors, reduced adult neurogenesis, accumulation of phosphorylated tau, and hippocampal atrophy with neuronal loss through an increased 4R-T/3R-T ratio. Normalization of this increased ratio by 4R-T-specific silencing results in recovery of the normal phenotype. These findings suggest a biological link among FUS/SFPQ, tau isoform alteration, and phenotypic expression, which may function in the early pathomechanism of FTLD.

FUS regulates AMPA receptor function and FTLD/ALS-associated behaviour via GluA1 mRNA stabilization.

FUS is an RNA/DNA-binding protein involved in multiple steps of gene expression and is associated with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). However, the specific disease-causing and/or modifying mechanism mediated by FUS is largely unknown. Here we evaluate intrinsic roles of FUS on synaptic functions and animal behaviours. We find that FUS depletion downregulates GluA1, a subunit of AMPA receptor. FUS binds GluA1 mRNA in the vicinity of the 3' terminus and controls poly (A) tail maintenance, thus regulating stability. GluA1 reduction upon FUS knockdown reduces miniature EPSC amplitude both in cultured neurons and in vivo. FUS knockdown in hippocampus attenuates dendritic spine maturation and causes behavioural aberrations including hyperactivity, disinhibition and social interaction defects, which are partly ameliorated by GluA1 reintroduction. These results highlight the pivotal role of FUS in regulating GluA1 mRNA stability, post-synaptic function and FTLD-like animal behaviours.

The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons.

TDP-43 and FUS are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and loss of function of either protein contributes to these neurodegenerative conditions. To elucidate the TDP-43- and FUS-regulated pathophysiological RNA metabolism cascades, we assessed the differential gene expression and alternative splicing profiles related to regulation by either TDP-43 or FUS in primary cortical neurons. These profiles overlapped by >25% with respect to gene expression and >9% with respect to alternative splicing. The shared downstream RNA targets of TDP-43 and FUS may form a common pathway in the neurodegenerative processes of ALS/FTLD.

Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

FUS-regulated region- and cell-type-specific transcriptome is associated with cell selectivity in ALS/FTLD.

FUS is genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To clarify the RNA metabolism cascade regulated by FUS in ALS/FTLD, we compared the FUS-regulated transcriptome profiles in different lineages of primary cells from the central nervous system. The profiles of FUS-mediated gene expression and alternative splicing in motor neurons were similar to those of cortical neurons, but not to those in cerebellar neurons despite the similarity of innate transcriptome signature. The gene expression profiles in glial cells were similar to those in motor and cortical neurons. We identified certain neurological diseases-associated genes, including Mapt, Stx1a, and Scn8a, among the profiles of gene expression and alternative splicing events regulated by FUS. Thus, FUS-regulated transcriptome profiles in each cell-type may determine cellular fate in association with FUS-mediated ALS/FTLD, and identified RNA targets for FUS could be therapeutic targets for ALS/FTLD.

Translational control of mitochondrial energy production mediates neuron morphogenesis.

Mitochondrial energy production is a tightly regulated process involving the coordinated transcription of several genes, catalysis of a plethora of posttranslational modifications, and the formation of very large molecular supercomplexes. The regulation of mitochondrial activity is particularly important for the brain, which is a high-energy-consuming organ that depends on oxidative phosphorylation to generate ATP. Here we show that brain mitochondrial ATP production is controlled by the cytoplasmic polyadenylation-induced translation of an mRNA encoding NDUFV2, a key mitochondrial protein. Knockout mice lacking the Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) have brain-specific dysfunctional mitochondria and reduced ATP levels, which is due to defective polyadenylation-induced translation of electron transport chain complex I protein NDUFV2 mRNA. This reduced ATP results in defective dendrite morphogenesis of hippocampal neurons both in vitro and in vivo. These and other results demonstrate that CPEB1 control of mitochondrial activity is essential for normal brain development.

Fine tuning of the redox function of Pseudomonas aeruginosa cytochrome c551 through structural properties of a polypeptide loop bearing an axial Met residue.

Pseudomonas aeruginosa cytochrome c(551) (PA) possesses a long polypeptide loop near its heme, and a unique hydrogen bond network among Ser52, axial Met61, and the heme 13-propionate side chain, i.e., Ser52 amide NH is hydrogen bonded to axial Met61 carbonyl CO, Met61 amide NH to Ser52 carbonyl CO, and Ser52 side chain OH to the heme 13-propionate side chain, contributes to stabilization of the structure of the loop [Y. Matsuura, T. Takano, R.E. Dickerson, J. Mol. Biol. 156 (1982) 389-409]. In this study, the structure and redox function of S52N and S52G mutants were characterized in order to elucidate the role of Ser52 in functional regulation of the protein. We found that the redox function of PA was hardly affected by an S52N mutation, but was slightly by an S52G one. The functional similarity between the wild-type protein and the S52N mutant demonstrated that Asn52 in the mutant plays a similar pivotal role in the formation of the unique hydrogen bond network that stabilizes the structure of the loop as Ser52 in the wild-type protein does. On the other hand, the functional alteration induced by the S52G mutation can be attributed to a structural change of the loop due to the lack of the hydrogen bond between the Gly52 and heme 13-propionate side chain in the mutant. Thus, this study demonstrated that the function of the protein can be tuned through the structural properties of the polypeptide loop near its heme.

The roles of stress-activated Sty1 and Gcn2 kinases and of the protooncoprotein homologue Int6/eIF3e in responses to endogenous oxidative stress during histidine starvation.

In fission yeast, Sty1 and Gcn2 are important protein kinases that regulate gene expression in response to amino acid starvation. The translation factor subunit Int6/eIF3e promotes Sty1-dependent response by increasing the abundance of Atf1, a transcription factor targeted by Sty1. While Gcn2 promotes expression of amino acid biosynthesis enzymes, the mechanism and function of Sty1 activation and Int6/eIF3e involvement during this nutrient stress are not understood. Here we show that mutants lacking sty1(+) or gcn2(+) display reduced viabilities during histidine depletion stress in a manner suppressible by the antioxidant N-acetyl cysteine, suggesting that these protein kinases function to alleviate endogenous oxidative damage generated during nutrient starvation. Int6/eIF3e also promotes cell viability by a mechanism involving the stimulation of Sty1 response to oxidative damage. In further support of these observations, microarray data suggest that, during histidine starvation, int6Δ increases the duration of Sty1-activated gene expression linked to oxidative stress due to the initial attenuation of Sty1-dependent transcription. Moreover, loss of gcn2 induces the expression of a new set of genes not activated in wild-type cells starved for histidine. These genes encode heatshock proteins, redox enzymes, and proteins involved in mitochondrial maintenance, in agreement with the idea that oxidative stress is imposed on gcn2Δ cells. Furthermore, early Sty1 activation promotes rapid Gcn2 activation on histidine starvation. These results suggest that Gcn2, Sty1, and Int6/eIF3e are functionally integrated and cooperate to respond to oxidative stress generated during histidine starvation.

Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.