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Continuous neuronal survival is vital for mammals because mammalian brains have limited regeneration capability. After neurogenesis, suppression of apoptosis is needed to ensure a neurons long-term survival. Here we describe a robust genetic program that intrinsically attenuates apoptosis competence in neurons. Developmental downregulation of the splicing regulator PTBP1 in immature neurons allows neural-specific splicing of the evolutionarily conserved Bak1 microexon 5. Exon 5 inclusion triggers nonsense-mediated mRNA decay (NMD) and unproductive translation of Bak1 transcripts (N-Bak mRNA), leading to suppression of pro-apoptotic BAK1 proteins and allowing neurons to reduce apoptosis. Germline heterozygous ablation of exon 5 increases BAK1 proteins exclusively in the brain, inflates neuronal apoptosis, and leads to early postnatal mortality. Therefore, neural-specific exon 5 splicing and depletion of BAK1 proteins uniquely repress neuronal apoptosis. Although apoptosis is important for development, attenuation of apoptosis competence through neural-specific splicing of the Bak1 microexon is essential for neuronal and animal survival.

The splicing regulator Polypyrimidine Tract Binding Protein (PTBP1) has four RNA binding domains that each binds a short pyrimidine element, allowing recognition of diverse pyrimidine-rich sequences. This variation makes it difficult to evaluate PTBP1 binding to particular sites based on sequence alone and thus to identify target RNAs. Conversely, transcriptome-wide binding assays such as CLIP identify many in vivo targets, but do not provide a quantitative assessment of binding and are informative only for the cells where the analysis is performed. A general method of predicting PTBP1 binding and possible targets in any cell type is needed. We developed computational models that predict the binding and splicing targets of PTBP1. A Hidden Markov Model (HMM), trained on CLIP-seq data, was used to score probable PTBP1 binding sites. Scores from this model are highly correlated (ρ = -0.9) with experimentally determined dissociation constants. Notably, we find that the protein is not strictly pyrimidine specific, as interspersed Guanosine residues are well tolerated within PTBP1 binding sites. This model identifies many previously unrecognized PTBP1 binding sites, and can score PTBP1 binding across the transcriptome in the absence of CLIP data. Using this model to examine the placement of PTBP1 binding sites in controlling splicing, we trained a multinomial logistic model on sets of PTBP1 regulated and unregulated exons. Applying this model to rank exons across the mouse transcriptome identifies known PTBP1 targets and many new exons that were confirmed as PTBP1-repressed by RT-PCR and RNA-seq after PTBP1 depletion. We find that PTBP1 dependent exons are diverse in structure and do not all fit previous descriptions of the placement of PTBP1 binding sites. Our study uncovers new features of RNA recognition and splicing regulation by PTBP1. This approach can be applied to other multi-RRM domain proteins to assess binding site degeneracy and multifactorial splicing regulation.

We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2(-/-) neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001.

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. We show here that central nervous system-specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1(-/-) brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.

A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.

PROM1 (CD133, AC133) is a protein that is required for the maintenance of primary cilia. Mutation in the Prom1 gene in humans and animal models are associated with several forms of retinal degeneration. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. We endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 protein. Deletion mutagenesis demonstrated that mAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1. The results of the mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. Our results show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has been fueled by SARS-CoV-2 novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strain's pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta-challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing challenged to no challenge mice revealed that Alpha-challenged mice have more total genes differentially activated. Conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-γ production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines. [ABSTRACT FROM AUTHOR]