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Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1–3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6–8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

SUMMARYEvolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1α, Ire1α, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1α in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1α engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1α in a mechanism of translational control dependent on 5’UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers.HIGHLIGHTSSmall molecule screening reveals Ire1α upstream of upper layer neuronal identityPolarization and proper lamination of layer II/III neurons require Ire1αDevelopment of upper layers requires high translation rates driven by eIF4A1 and eEF-2 downstream of Ire1αeIF4A1-dependent Satb2 mRNA translation initiation is a mechanism of upper layer fate acquisition

SUMMARYProtein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomesex vivofrom the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, within vivoembryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology.Graphical abstract

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the “neocortical ribosome signature.” Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.

On October 29, 2012, Hurricane Sandy made landfall in the neighborhood of Red Hook in Brooklyn, New York. The massive tidal surge generated by the storm submerged the coastal area, home to a population over 11,000 individuals, including the largest public housing development in Brooklyn. The infrastructure devastation was profound: the storm rendered electricity, heat, water, Internet, and phone services inoperative, whereas local ambulatory medical services including clinics, pharmacies, home health agencies, and other resources were damaged beyond functionality. Lacking these services or lines of communication, medically fragile individuals became isolated from the hospital and 911-emergency systems without a preexisting mechanism to identify or treat them. Medically fragile individuals primarily included those with chronic medical conditions dependent on frequent and consistent monitoring and treatments. In response, the Red Hook community established an ad hoc volunteer medical relief effort in the wake of the storm, filling a major gap that continues to exist in disaster medicine for low-income urban environments. Here we describe this effort, including an analysis of the medically vulnerable in this community, and recommend disaster risk reduction strategies and resilience measures for future disaster events. (Disaster Med Public Health Preparedness. 2015;9:354–358)

PIWI-interacting RNAs (piRNAs) and their associated PIWI proteins play an important role in repressing transposable elements in animal germlines. However, little is known about the function of PIWI proteins and piRNAs in the developing brain. Here, we investigated the role of an important PIWI family member, Piwi-like protein 1 (Piwil1; also known as Miwi in mouse) in the developing mouse neocortex. Using a Piwil1 knock-out (Piwil1 KO) mouse strain, we found that Piwil1 is essential for several steps of neocorticogenesis, including neocortical cell cycle, neuron migration and dendritogenesis. Piwil1 deletion resulted in increased cell cycle re-entry at embryonic day 17 (E17) when predominantly intracortically projecting neurons are being produced. Prenatal Piwil1 deletion increased the number of Pax6+ radial glia at postnatal day 0 (P0). Furthermore, Piwil1 deletion disrupted migration of Satb2+ neurons within deep layers at E17, P0 and P7. Satb2+ neurons showed increased co-localization with Bcl11b (also known as Ctip2), marker of subcortically projecting neurons. Piwil1 knockouts had disrupted neocortical circuitry represented by thinning of the corpus callosum and altered dendritogenesis. We further investigated if Piwil1 deletion disrupted expression levels of neocortical piRNAs by small RNA-sequencing in neocortex. We did not find differential expression of piRNAs in the neocortices of Piwil1 KO, while differences were observed in other Piwil1 KO tissues. This result suggests that Piwil1 may act independently of piRNAs and have novel roles in higher cognitive centers, such as neocortex. In addition, we report a screen of piRNAs derived from tRNA fragments in developing neocortices. Our result is the first report of selective subsets of piRNAs and tRNA fragments in developing prenatal neocortices and helps clarify some outstanding questions about the role of the piRNA pathway in the brain.

More than a passive effector of gene expression, mRNA translation (protein synthesis) by the ribosome is a rapidly tunable and dynamic molecular mechanism. Neurodevelopmental disorders are associated with abnormalities in mRNA translation, protein synthesis, and neocortical development; yet, we know little about the molecular mechanisms underlying these abnormalities. Furthermore, our understanding of regulation of the ribosome and mRNA translation during normal brain development is only in its early stages. mRNA translation is emerging as a key driver of the rapid and timed regulation of spatiotemporal gene expression in the developing nervous system, including the neocortex. In this review, we focus on the regulatory role of the ribosome in neocortical development, and construct a current understanding of how ribosomal complex specificity may contribute to the development of the neocortex. We also present a microarray analysis of ribosomal protein-coding mRNAs across the neurogenic phase of neocortical development, in addition to the dynamic enrichment of these mRNAs in actively translating neocortical polysomal ribosomes. Understanding the multivariate control of mRNA translation by ribosomal complex specificity will be critical to reveal the intricate mechanisms of normal brain development and pathologies of neurodevelopmental disorders.

Pre-mRNA alternative splicing is an important mechanism for the generation of synaptic protein diversity, but few factors governing this process have been identified. From a screen forDrosophilamutants with aberrant synaptic development, we identifiedbeag, a mutant with fewer synaptic boutons and decreased neurotransmitter release.Beagencodes a spliceosomal protein similar to splicing factors in humans andCaenorhabditis elegans. We find that bothbeagmutants and mutants of an interacting genedsmu1have changes in the synaptic levels of specific splice isoforms of Fasciclin II (FasII), theDrosophilaortholog of neural cell adhesion molecule. We show that restoration of one splice isoform of FasII can rescue synaptic morphology inbeagmutants while expression of other isoforms cannot. We further demonstrate that this FasII isoform has unique functions in synaptic development independent of transsynaptic adhesion.beaganddsmu1mutants demonstrate an essential role for these previously uncharacterized splicing factors in the regulation of synapse development and function.

Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA- binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

The mature neocortex is a unique six-layered mammalian brain region. It is composed of morphologically and functionally distinct subpopulations of primary projection neurons that form complex circuits across the central nervous system. The precisely-timed generation of projection neurons from neural stem cells governs their differentiation, postmitotic specification, and signaling, and is critical for cognitive and sensorimotor ability. Developmental perturbations to the birthdate, location, and connectivity of neocortical neurons are observed in neurological and psychiatric disorders. These facts are highlighting the importance of the precise spatiotemporal development of the neocortex regulated by intricate transcriptional, but also complex post-transcriptional events. Indeed, mRNA transcripts undergo many post-transcriptional regulatory steps before the production of functional proteins, which specify neocortical neural stem cells and subpopulations of neocortical neurons. Therefore, particular attention is paid to the differential post-transcriptional regulation of key transcripts by RNA-binding proteins, including splicing, localization, stability, and translation. We also present a transcriptome screen of candidate molecules associated with post-transcriptional mRNA processing that are differentially expressed at key developmental time points across neocortical prenatal neurogenesis.

Sažetak