Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

Accurate and precise annotation of 3' UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding protein-mediated mRNA 3' end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3' ends and compares favorably with existing 3' end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3' UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of clinical concern. In a cohort of 417 persons who had received the second dose of BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously, we identified 2 women with vaccine breakthrough infection. Despite evidence of vaccine efficacy in both women, symptoms of coronavirus disease 2019 developed, and they tested positive for SARS-CoV-2 by polymerase-chain-reaction testing. Viral sequencing revealed variants of likely clinical importance, including E484K in 1 woman and three mutations (T95I, del142-144, and D614G) in both. These observations indicate a potential risk of illness after successful vaccination and subsequent infection with variant virus, and they provide support for continued efforts to prevent and diagnose infection and to characterize variants in vaccinated persons. (Funded by the National Institutes of Health and others.).

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

Alzheimer's β-amyloid precursor protein (APP) associates with kinesin-1 via JNK-interacting protein 1 (JIP1); however, the role of JIP1 in APP transport by kinesin-1 in neurons remains unclear. We performed a quantitative analysis to understand the role of JIP1 in APP axonal transport. In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%). We identified two novel elements linked to JIP1 function, located in the central region of JIP1b, that interact with the coiled-coil domain of kinesin light chain 1 (KLC1), in addition to the conventional interaction of the JIP1b 11-amino acid C-terminal (C11) region with the tetratricopeptide repeat of KLC1. High frequency of APP anterograde transport is dependent on one of the novel elements in JIP1b. Fast velocity of APP cargo transport requires the C11 domain, which is regulated by the second novel region of JIP1b. Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK. Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b.

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

One of the best-characterized causative factors of Alzheimer's disease (AD) is the generation of amyloid-β peptide (Aβ). AD subjects are at high risk of epileptic seizures accompanied by aberrant neuronal excitability, which in itself enhances Aβ generation. However, the molecular linkage between epileptic seizures and Aβ generation in AD remains unclear.

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.

X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid β-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent.

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell type-specific post-transcriptional regulation has been a challenge. Here, we describe a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons.

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease (AD). Here, we investigated brain amyloid-β (Aβ) accumulation throughout the aging process in an amyloid precursor protein (APP) knock-in (KI) mouse model of AD that expresses human APPNL-G-F with or without human apoE4 or apoE3. Brain Aβ42 levels were significantly lower in 9-month-old mice that express human isoforms of apoE than in age-matched APP-KI control mice. Linear accumulation of Aβ42 began in 5-month-old apoE4 mice, and a strong increase in Aβ42 levels was observed in 21-month-old apoE3 mice. Aβ42 levels in cerebroventricular fluid were higher in apoE3 than in apoE4 mice at 6-7 months of age, suggesting that apoE3 is more efficient at clearing Aβ42 than apoE4 at these ages. However, apoE3 protein levels were lower than apoE4 protein levels in the brains of 21-month-old apoE3 and apoE4 mice, respectively, which may explain the rapid increase in brain Aβ42 burden in apoE3 mice. We identified genes that were downregulated in a human apoE-dependent (apoE4 > apoE3) and age-dependent (apoE3 = apoE4) manner, which may regulate brain Aβ burden and/or AD progression. Analysis of gene expression in AD mouse models helps identify molecular mechanisms of pleiotropy by the human APOE gene during aging.

Amyloid β (Aβ), a causative peptide of Alzheimer's disease, is generated by intracellular metabolism of amyloid β-protein precursor (APP). In general, mature APP (mAPP, N- and O-glycosylated form) is subject to successive cleavages by α- or β-, and γ-secretases in the late protein secretory pathway and/or at plasma membrane, while immature APP (imAPP, N-glycosylated form) locates in the early secretory pathway such as endoplasmic reticulum or cis-Golgi, in which imAPP is not subject to metabolic cleavages. X11-like (X11L) is a neural adaptor protein composed of a phosphotyrosine-binding (PTB) and two C-terminal PDZ domains. X11L suppresses amyloidogenic cleavage of mAPP by direct binding of X11L through its PTB domain, thereby generation of Aβ lowers. X11L expresses another function in the regulation of intracellular APP trafficking.

Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

We describe a patient haploinsufficient for the neuronal RNA binding protein NOVA1 who developed a behavioral motor hyperactivity disorder, suggesting a role of NOVA1 in postnatal motor inhibition. To investigate Nova1's action in adult Gad2+ inhibitory neurons, we generated a conditional Nova1-null mouse (Nova1-cKOGad2-cre). Strikingly, the phenotypes of these mice show many similarities to the NOVA1 haploinsufficient patient and identify a function of Nova1 in the hypothalamus. Molecularly, Nova1 loss in Gad2-positive neurons alters downstream expression of Impact mRNA, along with a subset of RNAs encoding electron transport chain-related factors and ribosomal proteins. NOVA1 stabilizes Impact mRNA by binding its 3' UTR, antagonizing the actions of miR-138 and miR-124. Together, these studies demonstrate actions of NOVA1 in adult hypothalamic neurons, mechanisms by which it functions in translation and metabolism, including through direct binding to Impact mRNA, and illuminate its role in human neurologic disease.

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establishes a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circEfnb2 was impaired when YCAY clusters located in flanking introns were mutagenized. CLIP (cross-linking and immunoprecipitation) and additional reporter analyses demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.