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.

Distinct developmental patterns in Anopheles stephensi organ systems.

Anatomical profiles of insects inform vector biology, comparative development and evolutionary studies with applications in forensics, agriculture and disease control. This study presents a comprehensive, high-resolution developmental profile of Anopheles stephensi, encompassing larval, pupal, and adult stages, obtained through microCT scanning. The results indicate in situ anatomical changes in most organ systems, including the central nervous system, eyes, musculature, alimentary canal, salivary glands, and ovaries, among other organ systems, except for the developing heart. We find significant differences in the mosquito gut, body-wall, and flight muscle development during metamorphosis from other dipterans like Drosophila. Specifically, indirect flight muscle specification and growth can be traced back at least to the 4th instar A. stephensi larvae, as opposed to post-puparial development in other Dipterans like Drosophila and Calliphora. Further, while Drosophila larval body-wall muscles and gut undergo histolysis, changes to these organs during mosquito metamorphosis are less pronounced. These observations, and raw data therein may serve as a reference for studies on the development and the genetics of mosquitoes. Overall, the detailed developmental profile of A. stephensi presented here illuminates the unique anatomy and developmental processes of Culicidae, with important implications for vector biology, disease control, and comparative evolutionary studies.

Eggs of the mosquito Aedes aegypti survive desiccation by rewiring their polyamine and lipid metabolism.

Upon water loss, some organisms pause their life cycles and escape death. While widespread in microbes, this is less common in animals. Aedes mosquitoes are vectors for viral diseases. Aedes eggs can survive dry environments, but molecular and cellular principles enabling egg survival through desiccation remain unknown. In this report, we find that Aedes aegypti eggs, in contrast to Anopheles stephensi, survive desiccation by acquiring desiccation tolerance at a late developmental stage. We uncover unique proteome and metabolic state changes in Aedes embryos during desiccation that reflect reduced central carbon metabolism, rewiring towards polyamine production, and enhanced lipid utilisation for energy and polyamine synthesis. Using inhibitors targeting these processes in blood-fed mosquitoes that lay eggs, we infer a two-step process of desiccation tolerance in Aedes eggs. The metabolic rewiring towards lipid breakdown and dependent polyamine accumulation confers resistance to desiccation. Furthermore, rapid lipid breakdown is required to fuel energetic requirements upon water reentry to enable larval hatching and survival upon rehydration. This study is fundamental to understanding Aedes embryo survival and in controlling the spread of these mosquitoes.

Local translation provides the asymmetric distribution of CaMKII required for associative memory formation.

How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

The transcriptional response to oxidative stress is independent of stress-granule formation.

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded protein and viral stress responses, whether and how SGs contribute to stress-induced transcription have not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3 h predominantly resulted in induction or up-regulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress transcriptome is enriched in mRNAs coding for chaperones including HSP70s, small heat shock proteins, glutathione transferases, and several noncoding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3 h after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

A C-terminal ataxin-2 disordered region promotes Huntingtin protein aggregation and neurodegeneration in Drosophila models of Huntington's disease.

The Ataxin-2 (Atx2) protein contributes to the progression of neurodegenerative phenotypes in animal models of amyotrophic lateral sclerosis (ALS), type 2 spinocerebellar ataxia (SCA-2), Parkinson's disease, and Huntington's disease (HD). However, because the Atx2 protein contains multiple separable activities, deeper understanding requires experiments to address the exact mechanisms by which Atx2 modulates neurodegeneration (ND) progression. Recent work on two ALS models, C9ORF72 and FUS, in Drosophila has shown that a C-terminal intrinsically disordered region (cIDR) of Atx2 protein, required for assembly of ribonucleoprotein (RNP) granules, is essential for the progression of neurodegenerative phenotypes as well as for accumulation of protein inclusions associated with these ALS models. Here, we show that the Atx2-cIDR also similarly contributes to the progression of degenerative phenotypes and accumulation of Huntingtin protein aggregates in Drosophila models of HD. Because Huntingtin is not an established component of RNP granules, these observations support a recently hypothesized, unexpected protein-handling function for RNP granules, which could contribute to the progression of Huntington's disease and, potentially, other proteinopathies.

Antagonistic roles for Ataxin-2 structured and disordered domains in RNP condensation.

Ataxin-2 (Atx2) is a translational control molecule mutated in spinocerebellar ataxia type II and amyotrophic lateral sclerosis. While intrinsically disordered domains (IDRs) of Atx2 facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using the Targets of RNA-Binding Proteins Identified by Editing technology, we identified an extensive data set of Atx2-target mRNAs in the Drosophila brain and S2 cells. Atx2 interactions with AU-rich elements in 3'UTRs appear to modulate stability/turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain deletions demonstrate that Atx2 (1) interacts closely with target mRNAs within mRNP granules, (2) contains distinct protein domains that drive or oppose RNP-granule assembly, and (3) has additional essential roles outside of mRNP granules. These findings increase the understanding of neuronal translational control mechanisms and inform strategies for Atx2-based interventions under development for neurodegenerative disease.

RNP-Granule Assembly via Ataxin-2 Disordered Domains Is Required for Long-Term Memory and Neurodegeneration.

Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). We demonstrate that Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo. Remarkably, ΔIDR mutants that lack neuronal RNP granules show normal animal development, survival, and fertility. However, they show defects in long-term memory formation/consolidation as well as in C9ORF72 dipeptide repeat or FUS-induced neurodegeneration. Together, our findings demonstrate (1) that higher-order mRNP assemblies contribute to long-term neuronal plasticity and memory, and (2) that a targeted reduction in RNP-granule formation efficiency can alleviate specific forms of neurodegeneration.

The Long 3'UTR mRNA of CaMKII Is Essential for Translation-Dependent Plasticity of Spontaneous Release in Drosophila melanogaster.

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-untranslated region (UTR) form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII-null larvae allows us to test its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process that requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release, which cannot be fulfilled by synaptic CaMKII from the other pool.SIGNIFICANCE STATEMENT As a regulator of synaptic development and plasticity, CaMKII has important roles in both normal and pathological function of the nervous system. CaMKII shows high conservation between Drosophila and humans, underscoring the usefulness of Drosophila in modeling its function. Drosophila CaMKII-null mutants remain viable throughout development, enabling morphological and electrophysiological characterization. Although the structure of the synapse is normal, maternally contributed CaMKII does not localize to synapses. Zygotic production of CaMKII mRNA with a long 3'-untranslated region is necessary for modulating spontaneous neurotransmission in an activity-dependent manner, but not for viability. These data argue that regulation of CaMKII localization and levels by local transcriptional processes is conserved. This is the first demonstration of distinct functions for Drosophila CaMKII mRNA variants.

The cytoplasmic capping complex assembles on adapter protein nck1 bound to the proline-rich C-terminus of Mammalian capping enzyme.

Cytoplasmic capping is catalyzed by a complex that contains capping enzyme (CE) and a kinase that converts RNA with a 5'-monophosphate end to a 5' diphosphate for subsequent addition of guanylic acid (GMP). We identify the proline-rich C-terminus as a new domain of CE that is required for its participation in cytoplasmic capping, and show the cytoplasmic capping complex assembles on Nck1, an adapter protein with functions in translation and tyrosine kinase signaling. Binding is specific to Nck1 and is independent of RNA. We show by sedimentation and gel filtration that Nck1 and CE are together in a larger complex, that the complex can assemble in vitro on recombinant Nck1, and Nck1 knockdown disrupts the integrity of the complex. CE and the 5' kinase are juxtaposed by binding to the adjacent domains of Nck1, and cap homeostasis is inhibited by Nck1 with inactivating mutations in each of these domains. These results identify a new domain of CE that is specific to its function in cytoplasmic capping, and a new role for Nck1 in regulating gene expression through its role as the scaffold for assembly of the cytoplasmic capping complex.

Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

Poly(A) polymerase-based poly(A) length assay.

mRNA polyadenylation functions in nuclear export, translation, and stability. We describe an efficient protocol designed to assess poly(A) tail length that is based on 3' tailing by yeast poly(A) polymerase and product analysis to single-nucleotide resolution by capillary electrophoresis.

Identification of cytoplasmic capping targets reveals a role for cap homeostasis in translation and mRNA stability.

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5' ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3' untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

Identification of the human PMR1 mRNA endonuclease as an alternatively processed product of the gene for peroxidasin-like protein.

The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The peroxidase genes arose from multiple duplication and rearrangement events, and their high degree of sequence similarity confounded attempts to identify human PMR1. The functioning of PMR1 in mRNA decay depends on the phosphorylation of a tyrosine in the C-terminal polysome targeting domain by c-Src. The sequences of regions that are required for c-Src binding and phosphorylation of Xenopus PMR1 were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here confirm the existence of the predicted 57-kDa protein, show this is the only form of PXDNL detected in any of the human cell lines examined, and confirm its identity as human PMR1. Like the Xenopus protein, human PMR1 binds to c-Src, is tyrosine phosphorylated, sediments on polysomes, and catalyzes the selective decay of a PMR1 substrate mRNA. Importantly, the expression of human PMR1 stimulates cell motility in a manner similar to that of the Xenopus PMR1 expressed in human cells, thus providing definitive evidence linking endonuclease decay to the regulation of cell motility.

Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b.

Contact of Mycobacterium tuberculosis (M.tb) with the immune system requires interactions between microbial surface molecules and host pattern recognition receptors. Major M.tb-exposed cell envelope molecules, such as lipomannan (LM), contain subtle structural variations that affect the nature of the immune response. Here we show that LM from virulent M.tb (TB-LM), but not from avirulent Myocobacterium smegmatis (SmegLM), is a potent inhibitor of TNF biosynthesis in human macrophages. This difference in response is not because of variation in Toll-like receptor 2-dependent activation of the signaling kinase MAPK p38. Rather, TB-LM stimulation leads to destabilization of TNF mRNA transcripts and subsequent failure to produce TNF protein. In contrast, SmegLM enhances MAPK-activated protein kinase 2 phosphorylation, which is critical for maintaining TNF mRNA stability in part by contributing microRNAs (miRNAs). In this context, human miRNA miR-125b binds to the 3' UTR region of TNF mRNA and destabilizes the transcript, whereas miR-155 enhances TNF production by increasing TNF mRNA half-life and limiting expression of SHIP1, a negative regulator of the PI3K/Akt pathway. We show that macrophages incubated with TB-LM and live M.tb induce high miR-125b expression and low miR-155 expression with correspondingly low TNF production. In contrast, SmegLM and live M. smegmatis induce high miR-155 expression and low miR-125b expression with high TNF production. Thus, we identify a unique cellular mechanism underlying the ability of a major M.tb cell wall component, TB-LM, to block TNF biosynthesis in human macrophages, thereby allowing M.tb to subvert host immunity and potentially increase its virulence.

Glucose-stimulated translation regulation of insulin by the 5' UTR-binding proteins.

Insulin is the key regulator of glucose homeostasis in mammals, and glucose-stimulated insulin biosynthesis is essential for maintaining glucose levels in a narrow range in mammals. Glucose specifically promotes the translation of insulin in pancreatic ß-islet, and the untranslated regions of insulin mRNA play a role in such regulation. Specific factors in the ß-islets bind to the insulin 5' UTR and regulate its translation. In the present study we identify protein-disulfide isomerase (PDI) as a key regulator of glucose-stimulated insulin biosynthesis. We show that both in vitro and in vivo PDI can specifically associate with the 5' UTR of insulin mRNA. Immunodepletion of PDI from the islet extract results in loss of glucose-stimulated translation indicating a critical role for PDI in insulin biosynthesis. Similarly, transient overexpression of PDI resulted in specific translation activation by glucose. We show that the RNA binding activity of PDI is mediated through PABP. PDI catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the insulin 5' UTR. We also show that glucose stimulation of the islets results in activation of a specific kinase that can phosphorylate PDI. These findings identify PDI and PABP as important players in glucose homeostasis.

Dense cataract and microphthalmia (dcm) in BALB/c mice is caused by mutations in the GJA8 locus.

A spontaneous mutation in BALB/c mice that causes congenital dense cataract and microphthalmia (dcm) was reported previously. This abnormality was found to be inheritable and the mode of inheritance indicated that this phenotype is due to mutation of an autosomal recessive gene. We performed genetic screen to identify the underlying mutations through linkage analysis with the dcm progenies of F(1) intercross. We identified the region of mutation on chromosome 3 and further mapping and sequence analysis identified the mutation in the GJA8 gene that encodes for connexin 50. The mutation represents a single nucleotide change at position 64 (G to C) that results in a change in the amino acid glycine to arginine at position 22 (G22R) and is identical to the mutation previously characterized as lop10. However, the phenotype of these mice differ from that of lop10 mice and since it is one of the very few genetic models with recessive pattern of inheritance, we propose that dcm mice can serve as a useful model for studying the dynamics and interaction of the gap junction formation in mouse eye development.

Novel splice variant of mouse insulin2 mRNA: implications for insulin expression.

Insulin is a secreted peptide that controls glucose homeostasis in mammals, and insulin biosynthesis is regulated by glucose at many levels. Rodent insulin is encoded by two non-allelic genes. We have identified a novel splice variant of the insulin2 gene in mice that constitutes about 75% of total insulin2 mRNA. The alternate splicing does not alter the ORF but reduces the 5'UTR by 12 bases. A reporter gene containing the novel short 5'UTR, is more efficiently expressed in cells, suggesting that alternative splicing of insulin mRNA in mice could result in an additional level of regulation in insulin biosynthesis.