Biology subjects

Burge, C.

Publications and source records attributed to Burge, C..

9 recordsLinked to original sources

bioRxiv2025

A splice-switching antisense oligonucleotide approach for pediatric genetic epilepsies

Summary ParagraphVariants in ion channel genes are common causes of pediatric epilepsy, often leading to intractable seizures, developmental delay and other comorbidities, which increases risk of death. Pathogenic variants in the SCN8A gene, which encodes a voltage-gated sodium channel critical for action potential generation in the brain, account for [~]1% of genetic epilepsies. The voltage sensor in SCN8A domain 1 is encoded by one of two developmentally-regulated mutually exclusive alternative exons, 5N and 5A. We observe that variants in these exons are more likely to cause infantile spasms, a severe seizure type, than variants elsewhere in SCN8A, and that some pathogenic variants affect exon 5 splicing, impacting patient phenotype. Molecular and evolutionary analyses implicate the exon sequences of these and other voltage-gated ion channel alternative exons in splicing regulation. We identified antisense oligonucleotides (ASOs) that shift splicing of SCN8A exon 5N to 5A or vice versa. These ASOs normalize neuronal activity in patient-derived iPSC neurons, and reduce seizures and motor impairment and extend lifespan in a new exon 5N mutant mouse model. Our results demonstrate that splice-switching ASOs can effectively reduce the expression of pathogenic isoforms and rescue both seizure and non-seizure phenotypes. Similar approaches should be applicable to pediatric genetic epilepsies caused by mutations in other ion channel alternative exons.

neuroscience↗

bioRxiv2024

Sequence determinants of intron-mediated enhancement learned from thousands of random introns

Spliceosomal introns are a ubiquitous feature of eukaryotic genes, whose presence often boosts the expression of their host gene, a phenomenon known as intron-mediated enhancement (IME). IME has been noted across diverse genes and organisms, but remains mysterious in many respects. For example, how does intron sequence affect the magnitude of IME? In this study, we performed a massively parallel reporter assay (MPRA) to assess the effect of varying intron sequence on gene expression in a high-throughput manner, in human cells, using tens of thousands of synthetic introns with natural splice sites and randomized internal sequence. We observe that most random introns splice efficiently and enhance gene expression as well as or better than fully natural introns. Nearly all introns stimulate gene expression [~]eight-fold above an intronless control, at both mRNA and protein levels, suggesting that the primary mechanism acts to increase mRNA levels. IME strength is positively associated with splicing efficiency and with the intronic content of poly-uridine stretches, which we confirm using reporter experiments. Together, this work elucidates sequence determinants of IME from tens of thousands of random introns, and confirms that enhancement of gene expression is a general property of splicing.

molecular biology↗

bioRxiv2024

Widespread naturally variable human exons aid genetic interpretation

Most mammalian genes undergo alternative splicing. The splicing of some exons has been acquired or lost in specific mammalian lineages, but differences in splicing within the human population are poorly characterized. Using GTEx tissue transcriptomes from 838 individuals, we identified 56,415 exons which are included in mRNAs in some individuals but entirely excluded from others, which we term "naturally variable exons" (NVEs). NVEs impact three quarters of protein-coding genes, occur at all population frequencies, and are often absent from reference annotations. NVEs are more abundant in genes depleted of genetic loss-of-function mutations and aid in the interpretation of causal genetic variants. Genetic variants modulate the splicing of many NVEs, and 5UTR and coding-region NVEs are often associated with increased and decreased gene expression, respectively. Together, our findings characterize abundant splicing variation in the human population, with implications for a range of human genetic analyses.

genetics↗

bioRxiv2023

An Interpretable Model of pre-mRNA Splicing for Animal and Plant Genes

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3 and 5 splice sites (SS), excises introns and ligates exons. SS recognition and pairing is often influenced by splicing regulatory factors (SRFs) that bind to splicing regulatory elements (SREs). Several families of sequence-specific SRFs are known to be similarly ancient. Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines new models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models the predict SS locations with 83-86% accuracy in fish, insects and plants, and about 70% in mammals. Learned SRE motifs include both known SRF binding motifs as well as novel motifs, and both classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals and a greater reliance on SREs in mammalian splicing, and increased reliance on intronic SREs in plant splicing.

bioinformatics↗

bioRxiv2023

Molecular and Cellular Context Influences SCN8A Variant Function

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

neuroscience↗

bioRxiv2023

Improved modeling of RNA-binding protein motifs in an interpretable neural model of RNA splicing

Sequence-specific RNA-binding proteins (RBPs) play central roles in splicing decisions, but their exact binding locations and activities are difficult to predict. Here, we describe a modular splicing architecture that leverages in vitro-derived RNA affinity models for 79 human RBPs and the annotated human genome to produce improved models of RBP binding and activity. Binding and activity are modeled by separate Motif and Aggregator components that can be mixed and matched, enforcing sparsity to improve interpretability. Standard affinity models yielded reasonable predictions, but substantial improvements resulted from using a new Adjusted Motif (AM) architecture. While maintaining accurate modeling of in vitro binding, training these AMs on the splicing task yielded improved predictions of binding sites in vivo and of splicing activity, using independent crosslinking and massively parallel splicing reporter assay data. The modular structure of our model enables improved generalizability to other species (insects, plants) and to exons of different evolutionary ages.

genomics↗

bioRxiv2022

RBPamp: Quantitative Modeling of Protein-RNA Interactions in vitro Predicts in vivo Binding

RNA-binding proteins (RBPs) control the processing and function of cellular transcripts to effect post-transcriptional gene regulation. Sequence-specific binding of RBPs to millions of synthetic RNAs has been probed in vitro by RNA Bind-n-Seq (RBNS). Here we describe RBPamp, a bio-physically-based model of protein-RNA interactions and associated algorithm that inferred affinity spectra of 79 diverse human RBPs from RBNS data. RBPamp supports multiple motifs per RBP, models RBP concentration and binding site saturation, and accounts for the effects of RNA secondary structure. RBPamp affinities along transcripts are predictive of in vivo binding, as measured by eCLIP density. For many RBPs, average local eCLIP density increases monotonically with predicted affinity, and the shape of this relationship can suggest free protein concentrations and potential cooperativity. Together, these analyses demonstrate a powerful integrative approach for the quantitative dissection of RBP function.

bioinformatics↗

bioRxiv2021

Widespread occurrence of hybrid internal-terminal exons in human transcriptomes

Alternative RNA processing is a major mechanism for diversifying the human transcriptome. Messenger RNA isoform differences are predominantly driven by alternative first exons, cassette internal exons and alternative last exons. Despite the importance of classifying exons to understand isoform structure, there is a lack of tools to look at isoform-specific exon usage using RNA-sequencing data. We recently observed that alternative transcription start sites often arise near annotated internal exons, creating "hybrid" exons that can be used as both first or internal exons. To investigate the creation of hybrid exons, we built the HIT (Hybrid-Internal-Terminal) exon pipeline that systematically classifies exons depending on their isoform-specific usage. Using a combination of junction reads coverage and probabilistic modeling, the HIT index identified thousands of hybrid first-internal and internal-last exons that were previously misclassified. Hybrid exons are enriched in long genes with at least ten internal exons, have longer flanking introns and strong splice sites. The usage of hybrid exons varies considerably across human tissues, but they are predominantly used in brain, testis and colon cells. Notably, genes involved in RNA splicing have the highest fraction of intra-tissue hybrid exons. Further, we found more than 100,000 inter-tissue hybrid exons that changed from internal to terminal exons across tissues. By developing the first method that can classify exons according to their isoform contexts, our findings demonstrate the existence of hybrid exons, expand the repertoire of tissue-specific terminal exons and uncover unexpected complexities of the human transcriptome.

bioinformatics↗

bioRxiv2020

Structural evolution of the tissue-specific U2AF2 paralog and alternative splicing factor LS2

The Drosophila melanogaster LS2 protein is a tissue-specific paralog of U2AF2 that mediates testis-specific alternative splicing. In order to understand the structural mechanisms underlying the distinct RNA binding specificity we determined the solution structures of the LS2 RNA recognition motif (RRM) domains and characterized their interaction with cis-regulatory guanosine-rich RNA motifs found in intron regions upstream of alternatively spliced exons. We show that the guanosine-rich RNA adopts a G quadruplex (G4) fold in vitro. The LS2 tandem RRMs adopt canonical RRM folds that are connected by a 38-residue linker that harbors a small helical motif 0. The LS2 RRM2 domain and the 0 helix in the interdomain linker mediate interactions with the G4 RNA. The functional importance of these unique molecular features in LS2 is validated by mutational analysis in vitro and RNA splicing assays in vivo. RNA sequencing data confirm the enrichment of G4-forming LS2 target motifs near LS2-affected exons. Our data indicate a role of G quadruplex structures as cis-regulatory motifs in introns for the regulation of alternative splicing, that engage non-canonical interactions with a tandem RRM protein. These results highlight the intriguing molecular evolution of a tissue-specific splicing factor from its conserved U2AF2 paralog as a result of (retro-) gene duplication in D. melanogaster.

biochemistry↗