Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

Despite the many approaches to study differential splicing from RNA-seq, many challenges remain unsolved, including computing capacity and sequencing depth requirements. Here we present SUPPA2, a new method that addresses these challenges, and enables streamlined analysis across multiple conditions taking into account biological variability. Using experimental and simulated data, we show that SUPPA2 achieves higher accuracy compared to other methods, especially at low sequencing depth and short read length. We use SUPPA2 to identify novel Transformer2-regulated exons, novel microexons induced during differentiation of bipolar neurons, and novel intron retention events during erythroblast differentiation.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1417-1) contains supplementary material, which is available to authorized users.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentrationdependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sexspecific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals.All protein-coding genes undergo pre-mRNA processing, and the large majority of these genes are alternatively spliced (1). Alternative exons can change many functional aspects of mRNAs and their encoded proteins. The best understood functions are stop codons or frameshifts that are introduced by 20 -35% of alternative exons, which often destine the altered mRNA to nonsense-mediated decay. Examples described in the literature show that alternative splicing regulates the binding properties, intracellular localization, enzymatic activity, protein stability, and post-translational modifications of a large number of proteins (reviewed in Ref.2). Thus, it appears that alternative pre-mRNA processing is a key mechanism regulating the gene expression of complex organisms by generating multiple mRNA isoforms, which encode functionally diverse proteins. Despite its importance, the exact mechanisms governing splice site selection are still poorly understood. In vertebrate systems, protein complexes assemble transiently on exons, and their interaction with the splicing machinery as well as RNA-RNA interactions between spliceosomal proteins and pre-mRNA determine whether an exon is included or skipped (reviewed in Refs. 3 and 4).When isolated from nuclei of mammalian cells, RNA polymerase II transcripts are found assembled in large ribonucleoprotein 21-MDa complexes, the supraspliceosome, composed of all five spliceosomal small nuclear ribonucleoproteins as well as additional proteins. The entire repertoire of nuclear pre-mRNAs, independent of their length or number of introns, is individually found assembled in supraspliceosomes (reviewed in Ref. 5). Structural studies revealed that the supraspliceosome is composed of four substructure...