High-throughput phenotypic screening for functionally characterizing alt. exons

Division Of Basic Sciences - Nci

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Abstract

Alterative pre-mRNA splicing greatly diversifies the human transcriptome expanding the coding potential of the genome. RNA sequencing profiling studies have revealed that almost all multi-exon human genes are subject to alternative splicing. Networks of timely coordinated alternative exons and introns are under elaborate regulatory control in different cell and tissue types and upon altering environmental cues generating diverse transcriptomic and proteomic landscapes that can support different cell fates or stress responses. It is estimated that over 10% of pathogenic mutations impact alternative splice site activation, and dysregulation of alternative splicing has been associated with complex diseases such as cancer and neurodevelopmental disorders. Major challenges in the alternative splicing field include i) the functional characterization of alternative exons, the overwhelming majority of which remain uncharacterized and, ii) understanding how splicing programs are remodeled in various cell types or environmental conditions. My research group is using a combination of functional genomics and focused molecular analyses to address these challenges as elaborated below. Project 1: High-throughput identification and characterization of phenotypically critical alternative exons Frame-preserving alternatively spliced exons that overlap coding sequences and are devoid of premature stop codons are translated and contribute to protein diversity. The functional consequences of such splice isoforms are difficult to predict, and it has been a matter of debate to what extent they represent stochastic differences in splice site selection during transcription with little biological repercussions9,10. Accordingly, a major challenge in the splicing field is to identify the full repertoire of biologically relevant alternative exons that contribute to phenotypic outcomes. Such knowledge will facilitate the prioritization of alternative splicing events for focused studies and will also provide predictive phenotypic insights of disease or disorder states characterized by widespread splicing alterations. To enable exon-resolution functional genomics, we have developed orthogonal ultra-efficient exon deletion and splice site mutation screening platforms and we have shown that can be used for the high-throughput interrogation phenotypically relevant exons in the human genome. Aim 1: Systematic identification of the human exons underlying cell fitness & proliferation. The first aim of our group has been to systematically profile the human exome to identify individual exons that can impact cell fitness and proliferation, a very important and quantitative cellular phenotype. To achieve this, we took advantage of the tools we have developed and generated guide (g)RNA libraries for exon deletion and splice site mutation screening to profile 15,000 frame-preserving exons from 2,500 genes in human HAP1 and RPE1 cells in total. Reassuringly, both screening strategies result in a significant overlap and positive correlation of exons that affect cell fitness. Our screens revealed that 20% of the interrogated exons affect cell fitness and these exons are more frequently found in highly expressed and essential genes. The cell fitness-affecting exons tend to be conserved in vertebrates and frequently overlap post-translation modification sites. Furthermore, we have shown that the fitness exons are enriched for overlapping modular protein domains and protein-protein interaction interfaces, while being devoid of low complexity regions. A manuscript describing these data is under revision. Aim 2: Development and application of exon-resolution genetic screening coupled to high-dimensional single-cell transcriptomic read-out. The transcriptome represents a very powerful and information-rich phenotype of cellular state and thus the coupling of CRISPR screens with single-cell RNA-Sequencing (scRNA-Seq) has been harnessed for examining high-dimensional phenotypic landscapes. To enable exon deletion screens with high-dimensional phenotypic profiling, we applied a series of optimization steps to have been able to combine exon deletion screens with single-cell RNA-Seq phenotypic readouts (an approach dubbed as scCHyMErA-Seq). We are currently performing a large-scale exon perturbation screen, targeting 500 exons for deletion, coupled to scRNA-Seq readouts. In these experiments, we built on our previously performed proliferation screens (see Aim 1), which identified 1,000 exons that affect cell fitness in human cells. We have generated CHyMErA libraries to excise a subset of these exons (with a focus on exons residing in gene expression regulators) and assess the gene expression changes in individual cells using scCHyMErA-Seq. This study represents the first large scale exon perturbation screening strategy with dimensional phenotypic read out and is fundamental novel insights of the exons in the human genome that affect gene expression signatures. Aim 3: In depth mechanistic characterization of alternative exons in transcription factors that control gene expression outputs. A major interest of our group is to functionally characterize alternative exons that can impact transcription, pre-mRNA processing or translation as demonstrated by our previous work. Both the cell fitness as well as the scCHyMErA screens have identified dozens of frame-preserving alternative exons in transcription factors. Focused follow-up studies of an alternative exon in the TFIID general transcription initiation factor component TAF5, uncovers that the inclusion levels of this single exon can control the overall assembly of the TFIID complex. Forced skipping of this alternatively spliced exon results in widespread gene expression alterations suggesting that this exon functions as a regulatory switch to tune TFIID formation and activity by controlling TATA-binding protein recruitment to transcription start sites. This study highlights the power of applying exon perturbation screens to interrogate phenotypically important exons at genome-scale, as a means for uncovering novel mechanisms that control gene expression and cell fitness. Preliminary analysis of our exon deletion single-cell RNA-Sequencing screens has revealed that among other exons, an alternative exon in NRF1, nuclear respiratory transcription factor 1, affects gene expression signatures. Based on these observations, we hypothesize that this exon is important, at least partially, for NRF1 function and currently performing focused studies to understand the role of this exon in regulating NRF1 activity and gene expression outputs.