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.

Long live the RNAs: The guardians of neuronal longevity?

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

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.

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.

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.

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.

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.

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.

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.

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.

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 unclear. 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 locus and binding diverse proteins to achieve X-chromosome inactivation (XCI). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment.

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.

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.

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.

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.

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.

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.

Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse.

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent.

BCseq: accurate single cell RNA-seq quantification with bias correction.

With rapid technical advances, single cell RNA-seq (scRNA-seq) has been used to detect cell subtypes exhibiting distinct gene expression profiles and to trace cell transitions in development and disease. However, the potential of scRNA-seq for new discoveries is constrained by the robustness of subsequent data analysis. Here we propose a robust model, BCseq (bias-corrected sequencing analysis), to accurately quantify gene expression from scRNA-seq.

High-Throughput Screening Method to Identify Alternative Splicing Regulators.

Misregulation of alternative pre-mRNA splicing contributes to various diseases. Understanding how alternative splicing is regulated paves the way to modulating or correcting molecular pathogenesis of the diseases. Alternative splicing is typically regulated by trans RNA binding proteins and their upstream modulators.

A Cell-Based High-Throughput Method for Identifying Modulators of Alternative Splicing.

Alternative splicing, a key regulatory process of gene expression, is controlled by trans-acting factors that recognize cis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise.