Shiba: A unified computational method for robust identification of differential RNA splicing across platforms.

Alternative pre-mRNA splicing (AS) is a fundamental regulatory process that generates transcript diversity and cell type variation. We developed Shiba, a robust method integrating transcript assembly, splicing event identification, read counting, and statistical analysis, to efficiently quantify exon splicing levels across various types of RNA-seq datasets. Compared to existing pipelines, Shiba excels in capturing both annotated and unannotated or cryptic differential splicing events with superior accuracy, sensitivity, and reproducibility. Furthermore, Shiba's unique consideration of junction read imbalance and exon-body read coverage reduces false positives, essential for downstream functional analyses. We have further developed scShiba for single-cell/nucleus (sc/sn) RNA-seq data, enabling the exploration of splicing variations in heterogeneous cell populations. Both simulated and real data demonstrate Shiba's robustness across multiple sample sizes, including n=1 datasets and individual cell clusters from scRNA-seq. Application of Shiba on single replicates of RNA-seq identified new AS-NMD targets, and scShiba on snRNA-seq revealed intricate temporal AS regulation in dopaminergic neurons. Both Shiba and scShiba are provided in Docker/Singularity containers and Snakemake pipeline, enhancing accessibility and reproducibility. The comprehensive capabilities of Shiba and scShiba allow systematic and robust quantification of alternative splicing events, laying a solid foundation for mechanistic exploration of functional complexity in RNA splicing.

Long live the RNAs: The guardians of neuronal longevity?

In a recent publication in Science, Zocher et al.1 identify and characterize long-lived nuclear RNA in the mouse brain, suggesting their potential roles as guardians of neuronal longevity.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

Alternative splicing in neurodegenerative disease and the promise of RNA therapies.

Alternative splicing generates a myriad of RNA products and protein isoforms of different functions from a single gene. Dysregulated alternative splicing has emerged as a new mechanism broadly implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer disease, amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson disease and repeat expansion diseases. Understanding the mechanisms and functional outcomes of abnormal splicing in neurological disorders is vital in developing effective therapies to treat mis-splicing pathology. In this Review, we discuss emerging research and evidence of the roles of alternative splicing defects in major neurodegenerative diseases and summarize the latest advances in RNA-based therapeutic strategies to target these disorders.

Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation.

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

Quantitative Measurement of Alternatively Spliced RNA Isoform Levels.

Conventional approaches to quantify alternative splicing are exon-centric and derive a ratio based on relative levels of the isoforms (or isoform groups) that include versus exclude a particular alternative RNA segment. The ratio measurement to study alternative splicing regulation can be confounded when alternative isoforms undergo differential RNA decay, for example, nonsense-mediated mRNA decay (NMD). Isoform-centric quantification is more informative for functional studies of alternative splicing, but challenges remain in distinguishing specific isoforms. Here, we provide a practical guide on addressing the specificity of isoform quantification and describe a simple sensitive method. Quantitative measurement of alternatively spliced RNA isoforms can be used to differentiate splicing regulation from transcriptional control and isoform-specific RNA decay regulation.

Genetic loss of function of Ptbp1 does not induce glia-to-neuron conversion in retina.

Direct reprogramming of glia into neurons is a potentially promising approach for the replacement of neurons lost to injury or neurodegenerative disorders. Knockdown of the polypyrimidine tract-binding protein Ptbp1 has been recently reported to induce efficient conversion of retinal Mϋller glia into functional neurons. Here, we use a combination of genetic lineage tracing, single-cell RNA sequencing (scRNA-seq), and electroretinogram analysis to show that selective induction of either heterozygous or homozygous loss-of-function mutants of Ptbp1 in adult retinal Mϋller glia does not lead to any detectable level of neuronal conversion. Only a few changes in gene expression are observed in Mϋller glia following Ptbp1 deletion, and glial identity is maintained. These findings highlight the importance of using genetic manipulation and lineage-tracing methods in studying cell-type conversion.

Multilayered regulations of alternative splicing, NMD, and protein stability control temporal induction and tissue-specific expression of TRIM46 during axon formation.

The gene regulation underlying axon formation and its exclusiveness to neurons remains elusive. TRIM46 is postulated to determine axonal fate. We show Trim46 mRNA is expressed before axonogenesis, but TRIM46 protein level is inhibited by alternative splicing of two cassette exons coupled separately to stability controls of Trim46 mRNA and proteins, effectively inducing functional knockout of TRIM46 proteins. Exon 8 inclusion causes nonsense-mediated mRNA decay of Trim46 transcripts. PTBP2-mediated exon 10 skipping produces transcripts encoding unstable TRIM46 proteins. During axonogenesis, transcriptional activation, decreased exon 8 inclusion, and enhanced exon 10 inclusion converge to increase TRIM46 proteins, leading to its neural-specific expression. Genetic deletion of these exons alters TRIM46 protein levels and shows TRIM46 is instructive though not always required for AnkG localization nor a determinant of AnkG density. Therefore, two concurrently but independently regulated alternative exons orchestrate the temporal induction and tissue-specific expression of TRIM46 proteins to mediate axon formation.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

Global Reprogramming of Apoptosis-Related Genes during Brain Development.

To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.

Understand variability of COVID-19 through population and tissue variations in expression of SARS-CoV-2 host genes.

An urgent question of coronavirus disease 2019 (COVID-19) is population variation in susceptibility to SARS-CoV-2 infection and symptom severity. We explore the expression profiles of SARS-CoV-2 host genes, their population variations, associated genetic variants, age- and sex-dependency in normal individuals. SARS-CoV-2 host genes are provisionally defined as the human genes that are experimentally validated or bioinformatically predicted to interact with SARS-CoV-2 proteins. Genes exhibiting most variable expression include ACE2, CLEC4G, CLEC4M, CD209 (interact with the SARS-CoV-2 spike protein); REEP6 (a receptor accessory protein expressed in the olfactory epithelium); SLC27A2 and PKP2 (inhibit virus replication); and PTGS2 (mediates fever response). SNP rs4804803, associated with SARS severity, affects expression of CLEC4G and CD209. Genetic variants of proteases associated with SARS-CoV-2 entry (TMPRSS2, CTSB, and CTSL) are strongly associated with their expression variation, suggesting a genetic contribution to phenotypic variations in multiple organs upon virus attack. The most significant age-dependent gene is ACE2, the cellular receptor of SARS-CoV-2. Others include TGF-ß family member GDF15, mediating inflammation, and VKORC1, possibly explaining vitamin K deficiency in COVID-19. TIMM10 and ERGIC1 exhibit significant sex differences. In summary, our results show genetic and multiple biological variables may underlie the population variation in SARS-CoV-2 infection and symptom severity.

Publisher Correction: A protein assembly mediates Xist localization and gene silencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A protein assembly mediates Xist localization and gene silencing.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3-5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

Developmental Attenuation of Neuronal Apoptosis by Neural-Specific Splicing of Bak1 Microexon.

Continuous neuronal survival is vital for mammals because mammalian brains have limited regeneration capability. After neurogenesis, suppression of apoptosis is needed to ensure a neuron's 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.

Putative Coiled-Coil Domain-Dependent Autoinhibition and Alternative Splicing Determine SHTN1's Actin-Binding Activity.

The actin cytoskeleton plays a pivotal role in cell development, morphogenesis, and other cellular functions. Precise control of actin dynamics requires actin-binding proteins. Here, we characterize multifarious regulation of SHTN1 (shootin1) and show that, unlike known actin-binding proteins, SHTN1's actin binding activity is intrinsically inhibited by a putative coiled-coil domain (CCD) and the autoinhibition is overcome by alternative splicing regulation. We found SHTN1 contains a noncanonical WH2 domain and an upstream proline-rich region (PRR) that by themselves are sufficient for actin interaction. Alternative splicing of Shtn1 at the C terminus and downstream of the WH2-PRR domain produces a long (SHTN1L or shootin1b) and a short (SHTN1S or shootin1a) isoform, which both contain the described PRR and WH2 domains. However, SHTN1S does not interact with actin due to inhibition mediated by an N-terminal CCD. A SHTN1L-specific C-terminal motif counters the intramolecular inhibition and allows SHNT1L to bind actin. A nuclear localization signal is embedded between PRR and WH2 and is subject to similar autoinhibition. SHTN1 would be the first WH2-containing molecule that adopts CCD-dependent autoinhibition and alternative splicing-dependent actin interaction.

Alternative splicing programming of axon formation.

Alternative pre-mRNA splicing generates multiple mRNA isoforms of different structures and functions from a single gene. While the prevalence of alternative splicing control is widely recognized, and the underlying regulatory mechanisms have long been studied, the physiological relevance and biological necessity for alternative splicing are only slowly being revealed. Significant inroads have been made in the brain, where alternative splicing regulation is particularly pervasive and conserved. Various aspects of brain development and function (from neurogenesis, neuronal migration, synaptogenesis, to the homeostasis of neuronal activity) involve alternative splicing regulation. Recent studies have begun to interrogate the possible role of alternative splicing in axon formation, a neuron-exclusive morphological and functional characteristic. We discuss how alternative splicing plays an instructive role in each step of axon formation. Converging genetic, molecular, and cellular evidence from studies of multiple alternative splicing regulators in different systems shows that a biological process as complicated and unique as axon formation requires highly coordinated and specific alternative splicing events. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Development.

Mechanisms of Neuronal Alternative Splicing and Strategies for Therapeutic Interventions.

Many cellular and physiological processes are coordinated by regulatory networks that produce a remarkable complexity of transcript isoforms. In the mammalian nervous system, alternative pre-mRNA splicing generates functionally distinct isoforms that play key roles in normal physiology, supporting development, plasticity, complex behaviors, and cognition. Neuronal splicing programs controlled by RNA-binding proteins, are influenced by chromatin modifications and can exhibit neuronal subtype specificity. As highlighted in recent publications, aberrant alternative splicing is a major contributor to disease phenotypes. Therefore, understanding the underlying mechanisms of alternative splicing regulation and identifying functional splicing isoforms with critical phenotypic roles are expected to provide a comprehensive resource for therapeutic development, as illuminated by recent successful interventions of spinal muscular atrophy. Here, we discuss the latest progress in the study of the emerging complexity of alternative splicing mechanisms in neurons, and how these findings inform new therapies to correct and control splicing defects.

Axonogenesis Is Coordinated by Neuron-Specific Alternative Splicing Programming and Splicing Regulator PTBP2.

How a neuron acquires an axon is a fundamental question. Piecemeal identification of many axonogenesis-related genes has been done, but coordinated regulation is unknown. Through unbiased transcriptome profiling of immature primary cortical neurons during early axon formation, we discovered an association between axonogenesis and neuron-specific alternative splicing. Known axonogenesis genes exhibit little expression alternation but widespread splicing changes. Axonogenesis-associated splicing is governed by RNA binding protein PTBP2, which is enriched in neurons and peaks around axonogenesis in the brain. Cortical depletion of PTBP2 prematurely induces axonogenesis-associated splicing, causes imbalanced expression of axonogenesis-associated isoforms, and specifically affects axon formation in vitro and in vivo. PTBP2-controlled axonogenesis-associated Shtn1 splicing determines SHTN1's capacity to regulate actin interaction, polymerization, and axon growth. Precocious Shtn1 isoform switch contributes to disorganized axon formation of Ptbp2-/- neurons. We conclude that PTBP2-orchestrated alternative splicing programming is required for robust generation of a single axon in mammals.

Developmental Xist induction is mediated by enhanced splicing.

X-inactive-specific transcript (Xist) is a long noncoding RNA (lncRNA) essential for inactivating one of the two X chromosomes in mammalian females. Random X chromosome inactivation is mediated by Xist RNA expressed from the inactive X chromosome. We found that Xist RNA is unspliced in naïve embryonic stem (ES) cells. Upon differentiation, Xist splicing becomes efficient across all exons independent of transcription, suggesting interdependent or coordinated removal of Xist introns. In female cells with mutated polypyrimidine tract binding protein 1 (Ptbp1), differentiation fails to substantially upregulate mature Xist RNA because of a defect in Xist splicing. We further found both Xist129 and XistCAS RNA are unspliced in Mus musculus 129SvJ/Mus castaneous (CAS) hybrid female ES cells. Upon differentiation, Xist129 exhibits a higher splicing efficiency than XistCAS, likely contributing to preferential inhibition of the X129 chromosome. Single cell analysis shows that the allelic choice of Xist splicing is linked to the inactive X chromosome. We conclude post-transcriptional control of Xist RNA splicing is an essential regulatory step of Xist induction. Our studies shed light on the developmental roles of splicing for nuclear-retained Xist lncRNA and suggest inefficient Xist splicing is an additional fail-safe mechanism to prevent Xist activity in ES cells.