A Global Regulatory Mechanism for Activating an Exon Network Required for Neurogenesis

“nSR100 Binds Intronic UGC Motifs Adjacent to the 30 Splice Site of Target Exons In Vivo To investigate whether nSR100 binds to the UGC, C/U-rich, and/ or other motifs, we used PAR-iCLIP, which employs incorporation of the photoreactive nucleoside analog 4-thiouridine in nascent RNA to increase crosslinking efficiency…”

“, 2009), which is thought to contribute to neural exon inclusion (Makeyev et al., 2007; Zheng et al., 2012), Ptbp2 expression is not sufficient for the activation of nSR100 target exons.”

“We generated a merged in vivo binding map (Licatalosi et al., 2008) displaying the mean, normalized distributions of nSR100 293T cell PARiCLIP crosslink sites within 400 nt windows surrounding the 157 conserved target exons, and a set of 400 control exons that are not nSR100 targets but that have a comparable PSI dist…”

“vertebrate nervous system, which possesses among the most complex yet highly conserved repertoires of regulated AS events (Barbosa-Morais et al., 2012; Merkin et al., 2012).”

“Transcriptome-wideprofilinghasuncovered vast repertoires of splice variants in metazoan species, many of which are highly conserved and regulated in a cell- and tissuespecific manner (Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010; Nilsen andGraveley, 2010).”

“Consistent with reports showing that Ptbp1 primarily functions to repress exon splicing (Boutz et al., 2007; Han et al., 2014; Llorian et al., 2010), knockdown of Ptbp1”

“Among the detected nSR100 interaction partners are the two subunits of the U2 snRNP auxiliary splicing factor, U2AF65/ U2AF2 and U2AF35/U2AF1, which bind directly to the polypyrimidine tract and 30 splice site AG dinucleotide, respectively (Merendino et al., 1999; Wu et al., 1999; Zamore et al., 1992; Zorio and Blumen…”

“, 1992; Zorio and Blumenthal, 1999), and promote U2 snRNP binding to the pre-mRNA branch site during spliceosome assembly (Ruskin et al., 1988).”

“…which regulate a discrete network of exons enriched in synaptic and axon guidance genes, bind clusters of YCAY motifs concentrated near 50 splice sites to promote exon inclusion and to YCAY clusters proximal to 30 splice sites to promote exon skipping (Licatalosi et al., 2008; Ule et al., 2006).”

“Ptbp1and itsparalogPtbp2bind intronicC/U-richelementsupstreamof regulatedexons to repress the inclusionof neural exons, yet Ptbp1 also promotes exon inclusion when binding to C/U-rich elements downstream of regulated exons (Licatalosi et al., 2012; Llorian et al., 2010; Xue et al., 2009).”

“…U2AF2 and U2AF35/U2AF1, which bind directly to the polypyrimidine tract and 30 splice site AG dinucleotide, respectively (Merendino et al., 1999; Wu et al., 1999; Zamore et al., 1992; Zorio and Blumenthal, 1999), and promote U2 snRNP binding to the pre-mRNA branch site during spliceosome…”

“PAR-iCLIP Experiments PAR-iCLIP experiments were performed as described previously (Huppertz et al., 2014) with anti-Flag, anti-U2af65, and anti-PTBP1 antibodies.”

“Moreover, SRm160/300 and other SR proteins have been shown to promote ESE-dependent splicing through interactions that are independent of those required for binding of U2AF65 to the polypyrimidine tract (Kan and Green, 1999; Li and Blencowe, 1999; Guth et al., 1999).”

“Loss of Ptbp1 facilitates the inclusion of a neural-specific exon (exon 10) in Ptbp2 transcripts that promotes Ptbp2 expression by preventing turnover of its transcripts by the nonsense-mediated mRNA decay pathway (Boutz et al., 2007; Makeyev et al., 2007; Spellman et al., 2007).”

“…sites is further influenced by the quality of the polypyrimidine tract; increased length and pyrimidine content of the polypyrimidine tract, and its proximity to the AG dinucleotide, all contribute to increased 30 splice site strength (Chiara et al., 1997; Merendino et al., 1999; Reed, 1989).”

“…sites is further influenced by the quality of the polypyrimidine tract; increased length and pyrimidine content of the polypyrimidine tract, and its proximity to the AG dinucleotide, all contribute to increased 30 splice site strength (Chiara et al., 1997; Merendino et al., 1999; Reed, 1989).”

“Consistent with reports showing that Ptbp1 primarily functions to repress exon splicing (Boutz et al., 2007; Han et al., 2014; Llorian et al., 2010), knockdown of Ptbp1”

“…function in transcriptional control of nervous system development, including the myocyte enhancer factor 2 (Mef2) family of transcription activators (Figures 1D and 1E), which are critical for the formation of cortical neuronal layers and synaptic plasticity (Akhtar et al., 2012; Li et al., 2008).”

“, 2009), which is thought to contribute to neural exon inclusion (Makeyev et al., 2007; Zheng et al., 2012), Ptbp2 expression is not sufficient for the activation of nSR100 target exons.”

“…which bind directly to the polypyrimidine tract and 30 splice site AG dinucleotide, respectively (Merendino et al., 1999; Wu et al., 1999; Zamore et al., 1992; Zorio and Blumenthal, 1999), and promote U2 snRNP binding to the pre-mRNA branch site during spliceosome assembly (Ruskin et al., 1988).”

“While nSR100 promotes the inclusion of an exon required for the neuronal expression of the Ptbp1 paralog, Ptbp2 (Calarco et al., 2009), which is thought to contribute to neural exon inclusion (Makeyev et al.”

“The relative expression levels of Ptbp1 and Ptbp2 governed by this regulatory circuit contribute to establishing neural-specific AS patterns (Boutz et al., 2007; Li et al., 2014; Licatalosi et al., 2012; Zheng et al., 2012).”

“Transcriptome-wideprofilinghasuncovered vast repertoires of splice variants in metazoan species, many of which are highly conserved and regulated in a cell- and tissuespecific manner (Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010; Nilsen andGraveley, 2010).”

“The relative expression levels of Ptbp1 and Ptbp2 governed by this regulatory circuit contribute to establishing neural-specific AS patterns (Boutz et al., 2007; Li et al., 2014; Licatalosi et al., 2012; Zheng et al., 2012).”

“Moreover, SRm160/300 and other SR proteins have been shown to promote ESE-dependent splicing through interactions that are independent of those required for binding of U2AF65 to the polypyrimidine tract (Kan and Green, 1999; Li and Blencowe, 1999; Guth et al., 1999).”

“…function in transcriptional control of nervous system development, including the myocyte enhancer factor 2 (Mef2) family of transcription activators (Figures 1D and 1E), which are critical for the formation of cortical neuronal layers and synaptic plasticity (Akhtar et al., 2012; Li et al., 2008).”

“Since the interaction partners are enriched in early spliceosomal components, we used native gel electrophoresis (Das and Reed, 1999) to investigate whether nSR100 enhances formation of the U2 snRNP-containing prespliceosomal A complex (Figure 4B).”

“In this model, a complex of the SR-related proteins SRm160 and SRm300, which we detect as nSR100 interacting-proteins, promotes splicing by forming cross-intron and cross-exon interactions involving U1 snRNP bound to the 50 splice site, U2 snRNP bound to the branch site, and SR family proteins bound to exonic splicing…”

“Recently, a mutation in the Bronx waltzer (bv) mouse, which causes deafness and balance defects, was mapped to the nSR100/Srrm4 locus and shown to disrupt AS of genes linked to secretion and neurotransmission that are expressed in the inner ear (Nakano et al., 2012).”

“…sites is further influenced by the quality of the polypyrimidine tract; increased length and pyrimidine content of the polypyrimidine tract, and its proximity to the AG dinucleotide, all contribute to increased 30 splice site strength (Chiara et al., 1997; Merendino et al., 1999; Reed, 1989).”

“Moreover, SRm160/300 and other SR proteins have been shown to promote ESE-dependent splicing through interactions that are independent of those required for binding of U2AF65 to the polypyrimidine tract (Kan and Green, 1999; Li and Blencowe, 1999; Guth et al., 1999).”

“Consistent with reports showing that Ptbp1 primarily functions to repress exon splicing (Boutz et al., 2007; Han et al., 2014; Llorian et al., 2010), knockdown of Ptbp1”

“Analysis of RNA-seq data generated from in vitro differentiation of mouse embryonic stem cells (ESCs) to cortical glutamatergic neuronal cells (Hubbard et al., 2013) also revealed dramatic changes in the expression patterns of nSR100 and Ptbp1, with nSR100 showing a marked increase from almost no expression in ESCs to…”

“Moreover, SRm160/300 and other SR proteins have been shown to promote ESE-dependent splicing through interactions that are independent of those required for binding of U2AF65 to the polypyrimidine tract (Kan and Green, 1999; Li and Blencowe, 1999; Guth et al., 1999).”

“…whether oASD brains exhibited differential patterns of alternative splicing (AS), a feature found in human ASD brains (Gandal et al., 2018; Irimia et al., 2014; Parikshak et al., 2016; Parras et al., 2018). rMATS analysis (Shen et al., 2014) identified 560 genes that showed different AS…”

“It is intriguing that RNPS1, an auxiliary EJC component, can repress inclusion of such cryptic microexons, whereas, on the other hand, it promotes splicing of canonical neural microexons (Gonatopoulos-Pournatzis et al., 2018).”

“, 2013)), or overexpression (nSR100 in human 293T cells (Raj et al., 2014)), and their associated controls, to identify AS exons and microexons whose inclusion levels are affected by experimental manipulation of the expression of the splicing factors.”