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Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5′ UTRs. Furthermore, 5′ UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5′ UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders., Translational regulation of isoforms in the developing nervous system is not well understood. Here, the authors report translational de-repression of RNA binding protein isoforms at their 5′UTRs in the neocortex and show the neurodevelopmental risk of post-transcriptional dysregulation.

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.

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.

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