Gene Regulation

We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or sequencing methods that are used to quantify gene expression and alternative splicing across multiple contexts. This information is used as input for state of the art methods for Graphical Model Selection in order to recover the structure of a composite network that simultaneously models exon co-regulation and their cognate regulators. Community structure detection and social network analysis methods are used to identify distinct modules and key actors within the network. As a proof of concept for our framework we studied the splicing regulatory network for Drosophila development using the publicly available modENCODE data. The final model offers a comprehensive view of the splicing circuitry that underlies fly development. Identified modules are associated with major developmental hallmarks including maternally loaded RNAs, onset of zygotic gene expression, transitions between life stages and sex differentiation. Within-module key actors include well-known developmental-specific splicing regulators from the literature while additional factors previously unassociated with developmental-specific splicing are also highlighted. Finally we analyze an extensive battery of Splicing Factor knock-down transcriptome data and demonstrate that our approach captures true regulatory relationships.

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with ...

Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellula...

Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and c...

We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or sequencing methods that are used to quantify gene expression and alternative splicing across multiple contexts. This information is used as input for state of the art methods for Graphical Model Selection in order to recover the structure of a composite network that simultaneously models exon co-regulation and their cognate regulators. Community structure detection and social network analysis methods are used to identify distinct modules and key actors within the network. As a proof of concept for our framework we studied the splicing regulatory network for Drosophila development using the publicly available modENCODE data. The final model offers a comprehensive view of the splicing circuitry that underlies fly development. Identified modules are associate...

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role ...

SummaryHow closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the RNA of 158,844 single cells from these organoids at six developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable ‘developed’ state at a rate similar to human retina development in vivo and the transcriptomes of organoid cell types converged towards the transcriptomes of adult peripheral retinal cell types. The expression of disease-associated genes was significantly cell-type specific in adult retina and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanis...