Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons

Abstract

Graphical Abstract Highlights d Genome-wide CRISPR-Cas9 screens for detection of alternative splicing regulators d Identification of 200 regulators of neuronal microexons often disrupted in autism d Diverse regulators include chromatin, protein turnover and RNA processing factors d A mechanism for the definition of neuronal microexons SUMMARYAlternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Chromatin-mediated alternative splicing regulates cocaine reward behavior

“…By combining de novo motif search and CLIP-seq data from neurons, we identified Srsf11 as a putative splice factor regulating splicing across treatments. Given the recently discovered role of Srsf11 in microexon expression in neurons (Gonatopoulos-Pournatzis et al, 2018), it will be interesting to analyze microexon expression and splicing in our datasets, especially following dCas9-Set2 driven splicing of Srsf11. Furthermore, we expect that additional splice factors are involved in cocaine driven alternative splicing, such as A2BP1, the motif for which was identified in our datasets and is enriched in spliced genes following investigator administered cocaine (Feng et al, 2014).…”

“…Alternative splicing is a key mechanism for gene regulation in brain. Aberrant splicing is implicated in myriad neurological diseases, such as autism (Gonatopoulos-Pournatzis et al, 2018;Quesnel-Vallières et al, 2016;Voineagu et al, 2011), Rett syndrome (Cheng et al, 2017;Kriaucionis and Bird, 2004;Li et al, 2016), Huntington's disease (Lin et al, 1993;Sathasivam et al, 2013;Wood, 2013), spinal muscular atrophy (Cartegni et al, 2006;Lorson et al, 1999;Parente and Corti, 2018;Xiong et al, 2015) and schizophrenia (Gandal et al, 2018;Glatt et al, 2011;Morikawa and Manabe, 2010;Nakata et al, 2009;Wu et al, 2012). However, this mechanism of gene regulation is understudied in neuropsychiatric diseases, including drug addiction.…”

“…We identified the splice factor, serine and arginine rich splicing factor 11 (Srsf11, also known as SRp54) (Gonatopoulos-Pournatzis et al, 2018), as highly enriched amongst both cocaine and Set2 spliced genes.…”

“…We biochemically validated these findings in a replicate cohort of HSV-Set2 or HSV-Set2(R195G) injected mice ( Figure 4G) and Set2transfected N2a cells ( Figure 4H), using qPCR and PCR methods to measure gene expression (Supplement figure 4F) and alternative splicing ( Figure 4G-H), respectively. PCR validation of alternative splicing was accomplished using a single pair of primers to amplify multiple isoforms; PSI was quantified as the relative abundance of each isoform in a single lane (Gonatopoulos-Pournatzis et al, 2018). Furthermore, both Set2 overexpression and cocaine SA caused differential enrichment of H3K36me3 at the Srsf11 splice junction ( Figure 4I).…”

Hypersilencing of SRRM4 suppresses basal microexon inclusion and promotes tumor growth across cancers

“…SRRM4 target exon PSI was correlated against the expression and methylation of a set of background genes, in order to determine the significance of SRRM4 correlation by a randomization test. Three different background sets were included: (1) a random set of genes (5000 in expression background, 500 in methylation background), (2) 425 genes that are GO-related to splicing, and (3) 233 genes that are reported to regulate microexons 54 .…”