Alternative polyadenylation determines the functional landscape of inverted Alu repeats.

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

LSM12 and ME31B/DDX6 Define Distinct Modes of Posttranscriptional Regulation by ATAXIN-2 Protein Complex in Drosophila Circadian Pacemaker Neurons.

ATAXIN-2 (ATX2) has been implicated in human neurodegenerative diseases, yet it remains elusive how ATX2 assembles specific protein complexes to execute its physiological roles. Here we employ the posttranscriptional co-activator function of Drosophila ATX2 to demonstrate that LSM12 and ME31B/DDX6 are two ATX2-associating factors crucial for sustaining circadian rhythms. LSM12 acts as a molecular adaptor for the recruitment of TWENTY-FOUR (TYF) to ATX2. The ATX2-LSM12-TYF complex thereby stimulates TYF-dependent translation of the rate-limiting clock gene period (per) to maintain 24 hr periodicity in circadian behaviors. In contrast, ATX2 contributes to NOT1-mediated gene silencing and associates with NOT1 in a ME31B/DDX6-dependent manner. The ME31B/DDX6-NOT1 complex does not affect PER translation but supports high-amplitude behavioral rhythms along with ATX2, indicating a PER-independent clock function of ATX2. Taken together, these data suggest that the ATX2 complex may switch distinct modes of posttranscriptional regulation through its associating factors to control circadian clocks and ATX2-related physiology.

Metabolic flux from the Krebs cycle to glutamate transmission tunes a neural brake on seizure onset.

Kohlschütter-Tönz syndrome (KTS) manifests as neurological dysfunctions, including early-onset seizures. Mutations in the citrate transporter SLC13A5 are associated with KTS, yet their underlying mechanisms remain elusive. Here, we report that a Drosophila SLC13A5 homolog, I'm not dead yet (Indy), constitutes a neurometabolic pathway that suppresses seizure. Loss of Indy function in glutamatergic neurons caused "bang-induced" seizure-like behaviors. In fact, glutamate biosynthesis from the citric acid cycle was limiting in Indy mutants for seizure-suppressing glutamate transmission. Oral administration of the rate-limiting α-ketoglutarate in the metabolic pathway rescued low glutamate levels in Indy mutants and ameliorated their seizure-like behaviors. This metabolic control of the seizure susceptibility was mapped to a pair of glutamatergic neurons, reversible by optogenetic controls of their activity, and further relayed onto fan-shaped body neurons via the ionotropic glutamate receptors. Accordingly, our findings reveal a micro-circuit that links neural metabolism to seizure, providing important clues to KTS-associated neurodevelopmental deficits.

The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms.

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.

Circadian gating of light-induced arousal in Drosophila sleep.

Circadian rhythms and sleep homeostasis constitute the two-process model for daily sleep regulation. However, evidence for circadian control of sleep-wake cycles has been relatively short since clock-less animals often show sleep behaviors quantitatively comparable to wild-type. Here we examine Drosophila sleep behaviors under different light-dark regimes and demonstrate that circadian clocks gate light-induced arousal. Genetic excitation of tyrosine decarboxylase 2 (TDC2)-expressing neurons suppressed sleep more evidently at night, causing nocturnal activity. The arousal effects were likely mediated in part by glutamate transmission from the octopaminergic neurons and substantially masked by light. Application of T12 cycles (6-h light: 6-h dark) further showed that the light-sensitive effects of TDC2 neurons depended on the time of the day. In particular, light-sensing via visual input pathway led to strong sleep suppression at subjective night, and such an effect disappeared in clock-less mutants. Transgenic mapping revealed that light-induced arousal and free-running behavioral rhythms require distinct groups of circadian pacemaker neurons. These results provide convincing evidence that circadian control of sleep is mediated by the dedicated clock neurons for light-induced arousal.

LSM12-EPAC1 defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient.

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin ß1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.

ZNF598 co-translationally titrates poly(GR) protein implicated in the pathogenesis of C9ORF72-associated ALS/FTD.

C9ORF72-derived dipeptide repeat proteins have emerged as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). However, the mechanisms underlying their expression are not fully understood. Here, we demonstrate that ZNF598, the rate-limiting factor for ribosome-associated quality control (RQC), co-translationally titrates the expression of C9ORF72-derived poly(GR) protein. A Drosophila genetic screen identified key RQC factors as potent modifiers of poly(GR)-induced neurodegeneration. ZNF598 overexpression in human neuroblastoma cells inhibited the nuclear accumulation of poly(GR) protein and decreased its cytotoxicity, whereas ZNF598 deletion had opposing effects. Poly(GR)-encoding sequences in the reporter RNAs caused translational stalling and generated ribosome-associated translation products, sharing molecular signatures with canonical RQC substrates. Furthermore, ZNF598 and listerin 1, the RQC E3 ubiquitin-protein ligase, promoted poly(GR) degradation via the ubiquitin-proteasome pathway. An ALS-relevant ZNF598R69C mutant displayed loss-of-function effects on poly(GR) expression, as well as on general RQC. Moreover, RQC function was impaired in C9-ALS patient-derived neurons, whereas lentiviral overexpression of ZNF598 lowered their poly(GR) expression and suppressed proapoptotic caspase-3 activation. Taken together, we propose that an adaptive nature of the RQC-relevant ZNF598 activity allows the co-translational surveillance to cope with the atypical expression of pathogenic poly(GR) protein, thereby acquiring a neuroprotective function in C9-ALS/FTD.

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins.

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.

mtIF3 is locally translated in axons and regulates mitochondrial translation for axonal growth.

BACKGROUND: The establishment and maintenance of functional neural connections relies on appropriate distribution and localization of mitochondria in neurites, as these organelles provide essential energy and metabolites. In particular, mitochondria are transported to axons and support local energy production to maintain energy-demanding neuronal processes including axon branching, growth, and regeneration. Additionally, local protein synthesis is required for structural and functional changes in axons, with nuclear-encoded mitochondrial mRNAs having been found localized in axons. However, it remains unclear whether these mRNAs are locally translated and whether the potential translated mitochondrial proteins are involved in the regulation of mitochondrial functions in axons. Here, we aim to further understand the purpose of such compartmentalization by focusing on the role of mitochondrial initiation factor 3 (mtIF3), whose nuclear-encoded transcripts have been shown to be present in axonal growth cones. RESULTS: We demonstrate that brain-derived neurotrophic factor (BDNF) induces local translation of mtIF3 mRNA in axonal growth cones. Subsequently, mtIF3 protein is translocated into axonal mitochondria and promotes mitochondrial translation as assessed by our newly developed bimolecular fluorescence complementation sensor for the assembly of mitochondrial ribosomes. We further show that BDNF-induced axonal growth requires mtIF3-dependent mitochondrial translation in distal axons. CONCLUSION: We describe a previously unknown function of mitochondrial initiation factor 3 (mtIF3) in axonal protein synthesis and development. These findings provide insight into the way neurons adaptively control mitochondrial physiology and axonal development via local mtIF3 translation.

Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons.

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington's disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis.

Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster.

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.

The novel gene twenty-four defines a critical translational step in the Drosophila clock.

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (PER) as well as more modest effects on Timeless (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock.

Suboptimal Mitochondrial Activity Facilitates Nuclear Heat Shock Responses for Proteostasis and Genome Stability.

Thermal stress induces dynamic changes in nuclear proteins and relevant physiology as a part of the heat shock response (HSR). However, how the nuclear HSR is fine-tuned for cellular homeostasis remains elusive. Here, we show that mitochondrial activity plays an important role in nuclear proteostasis and genome stability through two distinct HSR pathways. Mitochondrial ribosomal protein (MRP) depletion enhanced the nucleolar granule formation of HSP70 and ubiquitin during HSR while facilitating the recovery of damaged nuclear proteins and impaired nucleocytoplasmic transport. Treatment of the mitochondrial proton gradient uncoupler masked MRP-depletion effects, implicating oxidative phosphorylation in these nuclear HSRs. On the other hand, MRP depletion and a reactive oxygen species (ROS) scavenger non-additively decreased mitochondrial ROS generation during HSR, thereby protecting the nuclear genome from DNA damage. These results suggest that suboptimal mitochondrial activity sustains nuclear homeostasis under cellular stress, providing plausible evidence for optimal endosymbiotic evolution via mitochondria-to-nuclear communication.

The trinity of ribosome-associated quality control and stress signaling for proteostasis and neuronal physiology.

Translating ribosomes accompany co-translational regulation of nascent polypeptide chains, including subcellular targeting, protein folding, and covalent modifications. Ribosome-associated quality control (RQC) is a co-translational surveillance mechanism triggered by ribosomal collisions, an indication of atypical translation. The ribosome-associated E3 ligase ZNF598 ubiquitinates small subunit proteins at the stalled ribosomes. A series of RQC factors are then recruited to dissociate and triage aberrant translation intermediates. Regulatory ribosomal stalling may occur on endogenous transcripts for quality gene expression, whereas ribosomal collisions are more globally induced by ribotoxic stressors such as translation inhibitors, ribotoxins, and UV radiation. The latter are sensed by ribosome-associated kinases GCN2 and ZAKα, activating integrated stress response (ISR) and ribotoxic stress response (RSR), respectively. Hierarchical crosstalks among RQC, ISR, and RSR pathways are readily detectable since the collided ribosome is their common substrate for activation. Given the strong implications of RQC factors in neuronal physiology and neurological disorders, the interplay between RQC and ribosome-associated stress signaling may sustain proteostasis, adaptively determine cell fate, and contribute to neural pathogenesis. The elucidation of underlying molecular principles in relevant human diseases should thus provide unexplored therapeutic opportunities. [BMB Reports 2021; 54(9): 439-450].

Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila.

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication.

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

Functional role of CREB-binding protein in the circadian clock system of Drosophila melanogaster.

Rhythmic histone acetylation underlies the oscillating expression of clock genes in the mammalian circadian clock system. Cellular factors that contain histone acetyltransferase and histone deacetylase activity have been implicated in these processes by direct interactions with clock genes, but their functional relevance remains to be assessed by use of appropriate animal models. Here, using transgenic fly models, we show that CREB-binding protein (CBP) participates in the transcriptional regulation of the Drosophila CLOCK/CYCLE (dCLK/CYC) heterodimer. CBP knockdown in pigment dispersing factor-expressing cells lengthens the period of adult locomotor rhythm with the prolonged expression of period and timeless genes, while CBP overexpression in timeless-expressing cells causes arrhythmic circadian behaviors with the impaired expression of these dCLK/CYC-induced clock genes. In contrast to the mammalian circadian clock system, CBP overexpression attenuates the transcriptional activity of the dCLK/CYC heterodimer in cultured cells, possibly by targeting the PER-ARNT-SIM domain of dCLK. Our data suggest that the Drosophila circadian clock system has evolved a distinct mechanism to tightly regulate the robust transcriptional potency of the dCLK/CYC heterodimer.

The voltage-gated potassium channel Shaker promotes sleep via thermosensitive GABA transmission.

Genes and neural circuits coordinately regulate animal sleep. However, it remains elusive how these endogenous factors shape sleep upon environmental changes. Here, we demonstrate that Shaker (Sh)-expressing GABAergic neurons projecting onto dorsal fan-shaped body (dFSB) regulate temperature-adaptive sleep behaviors in Drosophila. Loss of Sh function suppressed sleep at low temperature whereas light and high temperature cooperatively gated Sh effects on sleep. Sh depletion in GABAergic neurons partially phenocopied Sh mutants. Furthermore, the ionotropic GABA receptor, Resistant to dieldrin (Rdl), in dFSB neurons acted downstream of Sh and antagonized its sleep-promoting effects. In fact, Rdl inhibited the intracellular cAMP signaling of constitutively active dopaminergic synapses onto dFSB at low temperature. High temperature silenced GABAergic synapses onto dFSB, thereby potentiating the wake-promoting dopamine transmission. We propose that temperature-dependent switching between these two synaptic transmission modalities may adaptively tune the neural property of dFSB neurons to temperature shifts and reorganize sleep architecture for animal fitness.

A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system.

Sleep behaviors are observed even in nematodes and arthropods, yet little is known about how sleep-regulatory mechanisms have emerged during evolution. Here, we report a sleep-like state in the cnidarian Hydra vulgaris with a primitive nervous organization. Hydra sleep was shaped by homeostasis and necessary for cell proliferation, but it lacked free-running circadian rhythms. Instead, we detected 4-hour rhythms that might be generated by ultradian oscillators underlying Hydra sleep. Microarray analysis in sleep-deprived Hydra revealed sleep-dependent expression of 212 genes, including cGMP-dependent protein kinase 1 (PRKG1) and ornithine aminotransferase. Sleep-promoting effects of melatonin, GABA, and PRKG1 were conserved in Hydra However, arousing dopamine unexpectedly induced Hydra sleep. Opposing effects of ornithine metabolism on sleep were also evident between Hydra and Drosophila, suggesting the evolutionary switch of their sleep-regulatory functions. Thus, sleep-relevant physiology and sleep-regulatory components may have already been acquired at molecular levels in a brain-less metazoan phylum and reprogrammed accordingly.