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PubMed2026

Spatial isoform sequencing at single-cell resolution reveals cell-type-specific spatial isoform variability in multiple brain cell types.

Spatial long-read technologies are increasingly common but usually lack single-cell resolution. This leaves unanswered whether spatially variable isoforms reflect variability within one cell type or differences in region-specific cell-type composition. Here, we developed Spl-ISO-Seq2 (500-nm resolution) and accompanying software, Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing of >450 million barcodes versus 80,000 previously. Applying this to the adult mouse brain, we compared differential isoform abundance between known regions and spatial isoform patterns independent of predefined regions. Both identified overlapping hits, for example, Rps24 in oligodendrocytes. For known Snap25 spatial isoform variation, we show that it occurs in excitatory neurons. The region-agnostic approach also uncovered patterns missed by region-based comparisons, for example, for Ighm. Notably, many spatial isoform signals are not driven by cell-type composition alone. Finally, our software is applicable to many spatial and single-cell protocols, demonstrating reproducibility between platforms (for example, Visium HD/Stereo-seq). Overall, our experimental/analytical methods enable a submicron-resolution-isoform view and open avenues for spatial isoform disease research.

Animals↗

PubMed2025

Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure.

BACKGROUND: Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete. METHODS: Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results. RESULTS: The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer mechanism that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ≈30% of cell type-specific genes were polyform, using multiple isoforms tailored to cell type-specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity in normal hearts. Comparisons of cardiomyocytes across conditions uncovered 379 genes with marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non-protein-coding biotypes. In contrast, cell state-specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure-associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment. CONCLUSIONS: We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas.

Humans↗

PubMed2026

Estimating protein isoform abundances with [Formula: see text].

A single gene can encode multiple versions of a protein, dubbed isoforms, with varying functionality. Cellular control of isoform abundances is critical for multiple aspects of biology and is only partially regulated by transcript levels. While long-read sequencing facilitates transcript quantification, quantifying the resulting protein isoforms on a large scale is a major challenge, complicating biological interpretation of transcript alterations. Standard "bottom up" mass spectrometry can assess only short portions of isoforms called peptides, and these peptides often map onto more than one isoform. We introduce [Formula: see text] (Protein isoform Abundance Quantification), a Bayesian method that leverages multiomic information from the peptidome and transcriptome to provide accurate estimates of isoform abundance even when peptide mapping is ambiguous. [Formula: see text] offers several advantages over existing methods in a unified framework. It provides uncertainty quantification, integrates multiomic information for improved accuracy, and provides a rigorous framework for hypothesis testing. Extensive simulations show that [Formula: see text] consistently outperforms competing methods in detecting differentially abundant protein isoforms and estimating their abundances. We use [Formula: see text] to investigate differences in isoform abundance levels between people with schizophrenia and control subjects, confirming a long-held hypothesis that levels of the C4A isoform of Complement Component 4 are increased in schizophrenia while C4B is not. These results demonstrate that [Formula: see text] can identify significant variations in isoform abundance levels not previously possible.

Protein Isoforms↗

PubMed2025

Perplexity as a Metric for Isoform Diversity in the Human Transcriptome.

Long-read sequencing (LRS) has revealed a far greater diversity of RNA isoforms than earlier technologies, increasing the critical need to determine which, and how many, isoforms per gene are biologically meaningful. To define the space of relevant isoforms from LRS, many existing analysis pipelines rely on arbitrary expression cutoffs, but a single threshold cannot accommodate the broad variability in isoform complexity across genes, cell-types, and disease states captured by LRS. To address this, we propose using perplexity-an interpretable measure derived from entropy-that quantifies the effective number of isoforms per gene based on the full, unfiltered isoform ratio distribution. Calculating perplexity for 124 ENCODE4 PacBio LRS datasets spanning 55 human cell types, we show that it provides intuitive assessments of isoform diversity and captures uncertainty across genes with varying complexity. Perplexity can be calculated at multiple gene regulatory levels-from transcript to protein-to compare how isoform diversity is reduced across stages of gene expression. On average, genes have an ORF-level perplexity of 2.1, indicating production of two distinct protein isoforms. We extended this analysis to evaluate expression variation across tissues and identified 4,593 ORFs across 3,102 genes with moderate to extreme tissue-specificity. We propose perplexity as a consistent, quantitative metric for interpreting isoform diversity across genes, cell types, and disease states. All results are compiled into a community resource to enable cross-study comparisons of novel isoforms.

Journal Article↗

PubMed2026

The Role of Small Segmental Duplications in Generating Identical Isoforms Through Alternative Splicing Sites.

Alternative splicing plays a crucial role in expanding proteomic diversity but can also generate identical isoforms under certain conditions. While mutually exclusive splicing of tandem exons has occasionally been reported to produce identical isoforms, the extent to which other splicing events contribute to this phenomenon remains unclear. In this study, we demonstrate that alternative 5' and 3' splice site selection can also lead to the formation of identical isoforms, providing an additional type of splicing event for functional redundancy in transcriptomes. To address this, we analyzed reference genome annotations from 15 plant species, including Arabidopsis thaliana and wheat (Triticum aestivum), obtained from the RefSeq database. Identical isoforms were computationally defined as transcripts with distinct exon-intron structures but identical coding sequences. Our analysis reveals that the majority of alternative 5' and 3' fragments originate from small segmental duplications, suggesting that sequence repetition within gene regions facilitates the emergence of such splicing patterns. We also observed differences in the annotated 5' UTRs of some identical isoforms. However, since the alternative splicing sites themselves were not located within UTRs, these differences may reflect annotation uncertainty rather than genuine AS-derived variation. Given that UTR predictions in reference databases are not always precise, such observations should be interpreted cautiously. Expression analysis using an isoform-specific k-mer approach confirmed that identical isoforms can be differentially regulated. These findings suggest that, beyond expanding protein diversity, alternative splicing can also generate redundant isoforms that are differentially expressed at the RNA level, indicating potential regulatory roles. By elucidating the structural and regulatory factors contributing to the formation and retention of identical isoforms, our study provides new insights into the evolutionary and functional significance of alternative splicing in plants.

Alternative Splicing↗

PubMed2025

Progesterone receptor isoform modulation via enhancer activation regulates progesterone signaling in endometrial stromal cells.

OBJECTIVE: To investigate enhancer-mediated regulation of progesterone receptor (PGR) isoforms, PGR-A and PGR-B, in human endometrial stromal cells, and to determine how isoform modulation shapes the progesterone-responsive transcriptome and cistrome relevant to endometrial function. DESIGN: A clustered regularly interspaced short palindromic repeats-based functional genomic screen was used to identify distal enhancers in telomerase-immortalized human endometrial stromal cells. Subsequent clustered regularly interspaced short palindromic repeats targeting of identified enhancers and the PGR promoter was used to modulate PGR isoform balance and assess functional consequences. SUBJECTS: None. EXPOSURE: Engineered endometrial stromal cells were treated with medroxyprogesterone acetate or vehicle. MAIN OUTCOME MEASURES: PGR isoform expression was assessed by western blot, the progesterone-responsive transcriptome was characterized by bulk ribonucleic acid sequencing, and the PGR cistrome was characterized by Cut&Run. RESULTS: Two distal PGR enhancers were identified in endometrial stromal cells located approximately 60 and 220 kb upstream of the PGR transcription start site. Clustered regularly interspaced short palindromic repeats-based activation of these enhancers upregulated both PGR-A and PGR-B, whereas promoter activation primarily upregulated PGR-B. Bulk ribonucleic acid sequencing revealed that shifting the PGR isoform balance altered the progesterone-regulated transcriptome: PGR-A/B-equivalent cells exhibited proinflammatory gene signatures, whereas PGR-B-dominant cells demonstrated suppression of inflammatory signaling and altered cell cycle programs. The PGR Cut&Run profiling revealed distinct genomic binding patterns associated with each isoform profile. Integration of the PGR cistrome with chromatin interaction maps suggested that these isoforms directly regulate distinct gene subsets involved in inflammation and fibrosis. Mechanistically, estrogen receptor alpha (ESR1) indirectly activated PGR-A expression, potentially through recruitment of Forkhead box protein O1 (FOXO1) at the distal enhancer, suggesting a noncanonical, enhancer-mediated mechanism of PGR regulation. CONCLUSIONS: Distal enhancers regulate the PGR isoform balance and shape the progesterone-responsive transcriptome in human endometrial stromal cells. This enhancer-mediated mechanism expands current models of PGR regulation beyond promoter-level control and may offer potential therapeutic targets to restore normal progesterone responsiveness in conditions marked by PGR isoform imbalance.

Humans↗

PubMed2025

IsoBayes: a Bayesian approach for single-isoform proteomics inference.

MOTIVATION: Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. RESULTS: Here, we introduce IsoBayes, a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: (i) infer the presence/absence of each protein isoform (via a posterior probability), (ii) estimate its abundance (and credible interval), and (iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. AVAILABILITY AND IMPLEMENTATION: IsoBayes is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.

Proteomics↗

PubMed2023

A systematic CRISPR screen reveals redundant and specific roles for Dscam1 isoform diversity in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule 1 (Dscam1) encodes 19,008 diverse ectodomain isoforms via the alternative splicing of exon 4, 6, and 9 clusters. However, whether individual isoforms or exon clusters have specific significance is unclear. Here, using phenotype-diversity correlation analysis, we reveal the redundant and specific roles of Dscam1 diversity in neuronal wiring. A series of deletion mutations were performed from the endogenous locus harboring exon 4, 6, or 9 clusters, reducing to 396 to 18,612 potential ectodomain isoforms. Of the 3 types of neurons assessed, dendrite self/non-self discrimination required a minimum number of isoforms (approximately 2,000), independent of exon clusters or isoforms. In contrast, normal axon patterning in the mushroom body and mechanosensory neurons requires many more isoforms that tend to associate with specific exon clusters or isoforms. We conclude that the role of the Dscam1 diversity in dendrite self/non-self discrimination is nonspecifically mediated by its isoform diversity. In contrast, a separate role requires variable domain- or isoform-related functions and is essential for other neurodevelopmental contexts, such as axonal growth and branching. Our findings shed new light on a general principle for the role of Dscam1 diversity in neuronal wiring.

Animals↗

PubMed2026

Beyond the gene: isoform diversity as a key contributor to human brain disorders.

The human brain exhibits exceptional transcriptomic complexity, with alternative splicing, promoter usage, and polyadenylation generating extensive transcript-isoform diversity. Isoform dysregulation is increasingly implicated in neurodevelopmental and psychiatric disorders (NPDs), yet the landscape, function, and genetic regulation of brain isoforms remain poorly understood due to limitations of short-read RNA sequencing. Advances in long-read sequencing (LR-seq) enable scalable full-length transcriptome profiling with single-cell and spatial resolution across developmental stages. Here, we review recent progress in isoform discovery, quantification, functional annotation, and genetic regulation, highlighting emerging links to human neurodevelopment and disease. LR-seq studies have uncovered tens of thousands of previously unannotated brain isoforms, with neuronal maturation characterized by increased exon inclusion and progressive 3' untranslated region (3' UTR) lengthening. Isoform-resolved genetic mapping outperforms gene-level analyses for NPD gene discovery and mechanistic interpretation. We argue that a shift from gene-centric to isoform-centric frameworks is essential to fully capture regulatory complexity in human neurogenetics. Together, these advances establish isoform diversity as a fundamental yet underappreciated axis of brain gene regulation and a key entry point for dissecting NPD biology.

Humans↗

PubMed2025

The modern expansion of Dscam1 isoform diversity in Drosophila is linked to fitness and immunity.

Drosophila melanogaster Down Syndrome cell adhesion molecule 1 (Dscam1) gene encodes 38,016 diverse cell surface receptor proteins via alternative splicing, which have both nervous and immune functions. However, it remains elusive why organisms have evolved such an astonishing diversity of isoforms. Here, we show that fitness and immunity properties have driven the modern evolution of Dscam1 isoform diversity. We assess multiple aspects of fly fitness in deletion mutants harboring exon 4, 6, or 9 clusters, respectively, reducing ectodomain isoform diversity stepwise from 18,612 to 396. All fitness-related traits generally improved as the potential number of isoforms increased; however, the magnitude of the changes varied remarkably in a variable cluster-specific manner. Correlation analysis revealed that fitness-related traits were much more sensitive to reductions in Dscam1 diversity compared to canonical neuronal self/non-self discrimination. We conclude that the role of Dscam1 isoforms in canonical neuronal self-avoidance and self/non-self discrimination is mediated by a small fraction of all isoforms (<1/10), whereas a separate role essential for other developmental contexts and resistances, likely in fitness and immunity, requires almost full isoform diversity. Thus, fitness and immunity properties, rather than canonical neuronal functions, are the dominant drivers during the modern diversification of the Dscam1 isoform. Our findings suggest that Dscam1 diversity is closely linked to adaptation and species diversification in arthropods.

Animals↗

PubMed2025

Bayesian identification of differentially expressed isoforms using a novel joint model of RNA-seq data.

We develop a Bayesian approach, BayesIso, to identify differentially expressed isoforms from RNA-seq data. The approach features a novel joint model of the sample variability and the deferential state of isoforms. Specifically, the within-sample variability and the between-sample variability of each isoform are modeled by a Poisson-Lognormal model and a Gamma-Gamma model, respectively. Using a Bayesian framework, the differential state of each isoform and the model parameters are jointly estimated by a Markov Chain Monte Carlo (MCMC) method. Extensive studies using simulation and real data demonstrate that BayesIso can effectively detect isoforms of less differentially expressed and differential transcripts for genes with multiple isoforms. We applied the approach to breast cancer RNA-seq data and uncovered a unique set of isoforms that form key pathways associated with breast cancer recurrence. First, PI3K/AKT/mTOR signaling and PTEN signaling pathways are identified as being involved in breast cancer development. Further integrated with protein-protein interaction data, pathways of Jak-STAT, mTOR, MAPK and Wnt signaling are revealed in association with breast cancer recurrence. Finally, several pathways are activated in the early recurrence of breast cancer. In tumors that occur early, members of pathways of cellular metabolism and cell cycle (such as CD36 and TOP2A) are upregulated, while immune response genes such as NFATC1 are downregulated.

Humans↗

PubMed2026

k-mer-based Upstream Preprocessing of long reads for Isoform Discovery.

Eukaryotic genes can encode multiple protein isoforms based on alternative splicing of their transcribed regions. Most modern novel isoform discovery methods function by identifying and assembling exon splice junctions from an RNA-seq sample. However, splice junctions can only be accurately annotated with time-intensive dynamic programming alignment. This manuscript introduces KuPID, a method for preprocessing long RNA-seq reads with the goal of better identifying novel isoform transcripts. KuPID utilizes k-mer sketching as a prefilter to quickly pseudo-align reads to known reference isoforms. Full alignment need only then be applied to reads that are most relevant to isoform discovery. Not only does KuPID speed up the discovery pipeline, it also increases downstream accuracy by filtering out extraneous reads. KuPID preprocessing simultaneously increases the f1 accuracy of isoform discovery pipelines by up to 11.6 points while decreasing the runtime by a factor of 2-3×;. An optional mode permits a KuPID sample to be paired with both isoform discovery and transcript quantification.

Journal Article↗

PubMed2026

Interleukin-23 Receptor and Interleukin-17 Receptor A: Splice Variants, Isoforms and Their Relationship With Periodontitis-A Systematic Review and Bioinformatic Analysis.

This systematic review aimed to: (1) identify the splicing variants of IL23R and IL17RA reported in the literature; (2) perform a multiple alignment analysis to describe the isoforms of IL-23R and IL-17RA; and (3) compare the expression levels of IL-23R, IL-17RA, and their soluble isoforms (sIL-23R and sIL-17RA) in patients with periodontitis and periodontally healthy individuals. The study protocol followed PRISMA guidelines and was registered in PROSPERO (CRD420251267367). Six databases (PubMed, ScienceDirect, Scopus, Web of Science, EBSCO, and Google Scholar) were searched without restrictions on year or language. The descriptors used were: 'Interleukin-23 Receptor,' 'IL-23R,' 'Interleukin-17 Receptor A' 'IL-17RA,' 'Alternative Splicing,' 'Splice Variants,' 'Isoforms,' and 'Periodontitis.' The bioinformatics analysis was performed using CLUSTALW (V.1.83), InterPro and DeepTMHMM. Risk of bias was assessed with the QUIN and JBI tools for cross-sectional studies. Of 104 articles, four in vitro studies and eight cross-sectional studies were included. Qualitative analysis revealed that to date there are 32 splicing variants of the IL23R gene, while only one splicing variant has been reported for IL17RA. CLUSTALW, InterPro and DeepTMHMM analysis showed that these splicing variants result in 23 isoforms which can be soluble forms, complete intracellular peptides, truncated extracellular or intracellular peptides, or complete structures with truncated extracellular and/or intracellular domains. All studies had a low risk of bias. IL-23R and IL-17RA exhibit structural diversity resulting from alternative splicing, with IL-23R demonstrating significantly greater isoform complexity. However, the biological significance of these isoforms in periodontitis remains unclear and requires further investigation.

Humans↗

PubMed2026

Whole-transcriptome-scale isoform-resolved spatial imaging of single cells in tissues.

Cell and tissue functions arise from complex interactions among numerous genes, and a systematic understanding of these functions requires isoform-resolved transcriptomic analysis of single cells with high spatial resolution. Here, we introduce an in situ RNA amplification method and its integration with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to detect short RNA sequences and enable whole-transcriptome-scale, isoform-resolved spatial transcriptomics of individual cells in intact tissues. Using this approach, we imaged ∼33,000 distinct RNAs-including ∼23,000 genes and ∼10,000 isoforms-in the mouse brain. Our data enabled systematic analyses of region- and cell-type-specific gene programs and ligand-receptor-based cell-cell communications. These data further revealed rich spatial diversity and cell-type specificity in isoform usage across numerous genes, as well as brain structures particularly rich in isoform specificity. We anticipate broad application of this method for characterizing the molecular and cellular basis of tissue functions, unlocking previously inaccessible discoveries in cell and organismal biology.

Animals↗

PubMed2026

Isoform-Level Analysis Reveals Reproducible Early Changes in Transcript Usage During Human Vaccine Responses.

Vaccine-induced transcriptional responses have been extensively characterized at the gene level, but whether vaccination also alters transcript isoform usage remains largely unexplored. Here, we reanalyzed longitudinal whole-blood RNA-seq data from a discovery cohort of mRNA COVID-19 vaccine recipients using the IsoformSwitchAnalyzeR framework and validated the findings in an independent cohort. Key findings were validated by full-length RNA long-read sequencing and extended to four additional vaccine cohorts covering distinct platforms and pathogens. mRNA vaccination induced a rapid and transient wave of differential transcript usage, peaking at 24 h post-vaccination with 131 isoforms significantly altered across 107 genes, before largely resolving by Day 14. Isoform switching events were reproducible across independent cohorts and confirmed by full-length RNA long-read sequencing. Structural annotation of switching transcripts, including RMI2, WARS1, and NT5C3A, revealed changes affecting predicted protein domains and signal peptides. Notably, highly concordant isoform switching patterns were observed across MVA-based SARS-CoV-2, influenza, and Ebola vaccine cohorts and showed dose-dependent modulation. Overall, differential transcript isoform usage is a rapid and transient feature of the early human immune response to vaccination that was observed across multiple vaccine platforms. These findings reveal an underappreciated layer of transcriptional regulation that complements conventional gene-level analyses and warrants integration into future vaccine immunogenicity studies.

Humans↗

PubMed2025

iSoMAs: Finding isoform expression and somatic mutation associations in human cancers.

Aberrant alternative splicing, prevalent in cancer, impacts various cancer hallmarks involving proliferation, angiogenesis, and invasion. Splicing disruption often results from somatic point mutations rewiring functional pathways to support cancer cell survival. We introduce iSoMAs (iSoform expression and somatic Mutation Association), an efficient computational pipeline leveraging principal component analysis technique, to explore how somatic mutations influence transcriptome-wide gene expression at the isoform level. Applying iSoMAs to 33 cancer types comprising 9,738 tumor samples in The Cancer Genome Atlas, we identified 908 somatically mutated genes significantly associated with altered isoform expression across three or more cancer types. Mutations linked to differential isoform expression occurred through both cis- and trans-acting mechanisms, involving well-known oncogenes/suppressor genes, RNA binding protein and splicing factor genes. With wet-lab experiments, we verified direct association between TP53 mutations and differential isoform expression in cell cycle genes. Additional iSoMAs genes have been validated in the literature with independent cohorts and/or methods. Despite the complexity of cancer, iSoMAs attains computational efficiency via dimension reduction strategy and reveals critical associations between regulatory factors and transcriptional landscapes.

Humans↗

PubMed2026

Developmental analysis of the cone photoreceptor-less little skate retina reveals distinct Onecut1 isoforms.

The retinal development of elasmobranchs, the subclass comprising sharks, skates, and rays, remains poorly understood. This group is diverse in retinal phenotype, with many sharks and rays possessing rods together with one or more cone types. In contrast, the little skate (Leucoraja erinacea) has only a single rod photoreceptor type, which has been reported to exhibit some physiological and anatomical properties associated with cones. To investigate how this unusual photoreceptor system develops, we first identified an embryonic stage of early photoreceptor formation based on otx2 expression. We then developed a retinal electroporation approach to test whether a onecut1-dependent cone-associated reporter could be activated in the embryonic skate retina. Activation of this reporter was not detected, indicating that the corresponding enhancer is not robustly active under the conditions tested. To assess developmental changes in gene expression, we generated bulk RNA-seq datasets from embryonic, hatchling, and adult retinas. These analyses showed strong embryonic expression of onecut1, increasing expression of rod-associated genes through development, and pseudogenization or loss of multiple cone-enriched genes. We further identified a developmentally regulated onecut1 splice isoform containing an additional 48 amino acid sequence between the CUT and homeodomain DNA-binding domains. This spacer-containing isoform, termed LSOC1X2, was most abundant in the embryonic retina. To test whether LSOC1X2 retained regulatory activity, we assayed it in a mouse retinal reporter system. Both skate Onecut1 isoforms activated the ThrbCRM1 reporter in this heterologous context. Together, these findings identify a novel, developmentally regulated retinal onecut1 isoform in the little skate and establish it as a candidate regulator for future studies of photoreceptor development in this species and its elasmobranch relatives.

Animals↗

PubMed2026

Long-read proteogenomic atlas of human neuronal differentiation reveals isoform diversity informing neurodevelopmental risk mechanisms.

RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in induced pluripotent stem cell-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that autism risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between alternative transcript processing events, including transcription start sites, exon splicing, and polyadenylation. Finally, our atlas enables variant reinterpretation in autism, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment.

Humans↗