A Highly Conserved Program of Neuronal Microexons Is Misregulated in Autistic Brains

“3 (Shannon et al., 2003) with the following parameters: pvalue cut-off = 0.”

“These 50-nt split reads were then mapped to the corresponding genome assemblies using Bowtie (Langmead et al., 2009), using –m 1 –v 2 parameters (unique mapping with no more than two mismatches).”

“For gene-level differential expression analysis, reads were aligned to the human genome (hg19) with TopHat2 (Kim et al., 2013) and quantified with HT-seq Counts using a union-exon model from Gencode v18 (Anders et al.”

“, 2004); Transmembrane predictions, TMHMM (Krogh et al., 2001); ELM (Dinkel et al.”

“the empirical Bayes log-odds of differential PSI/PSU/PIRs (Smyth, 2004) (as implemented in “ebayes”, from the limma package in R).”

“, 2014); SignalP (Petersen et al., 2011); COIL (Lupas et al.”

“For example, transcripts from over 95% of human multiexon genes undergo AS, and most of the resulting mRNA splice variants are variably expressed between different cell and tissue types (Pan et al., 2008; Wang et al., 2008).”

“To further investigate the cell- and tissue-type specificity of microexon regulation, we used RNA-seq data (Sofueva et al., 2013; Zhang et al., 2013, 2014) to compare their inclusion levels in major glial cell types (astrocytes, microglia, and oligodendrocytes), in isolated neurons, and in muscle…”

“, 2013) databases with the dashed lines representing indirect interactions that are either between proteins found within same protein complex but not interacting directly or predicted interactions based on paralogs (Franceschini et al., 2013).”

“Next, we modeled the structure of microexon-containing Ensembl proteins using the Phyre fold-recognition server (Kelley and Sternberg, 2009), with default parameters.”

“To investigate the conservation of exonic sequences and their flanking intronic regions (150 nucleotides upstream and downstream of the exon), we used human phastCons (Siepel et al., 2005) data from alignments of 46 placental mammals (46way.”

“For example, transcripts from over 95% of human multiexon genes undergo AS, and most of the resulting mRNA splice variants are variably expressed between different cell and tissue types (Pan et al., 2008; Wang et al., 2008).”

“We thus used the Dali server (Holm and Rosenström, 2010) to identify structures that have been resolved in the presence of a protein ligand and that most closely resembles the APBB1-PTB1 domain.”

“These events are regulated by the interplay of cisacting motifs and trans-acting factors that control the assembly of spliceosomes (Chen andManley, 2009; Wahl et al., 2009).”

“In particular, microexons (Beachy et al., 1985; Coleman et al., 1987), defined here as 3–27 nucleotide (nt)-long exons, have been largely missed by genome annotations and transcriptome profiling studies (Volfovsky et al., 2003; Wu et al., 2013; Wu and Watanabe, 2005).”

“Gene Ontology analyses The Gene Ontology (GO) network for the human genes harboring neural-regulated AS events predicted to generate alternative protein isoforms (Figure 1C) was built using Enrichment Map (Merico et al., 2010) in Cytoscape 2.”

“Human disease mutations often disrupt cis-elements that control splicing and result in aberrant AS patterns (Cartegni et al., 2002).”

“, 2005), Disopred2 (Ward et al., 2004); CAST (Promponas et al.”

“, 2014) and, when necessary, SWISS-MODEL database (Kiefer et al., 2009), were calculated using the PISA software, as contained in version 6.”

“Taken together with data in Figures 5 and 6, the results suggest that themisregulation ofmicroexons, as well as of longer alternative exons (Corominas et al., 2014; Voineagu et al., 2011), may impact protein-interaction networks that are required for normal neuronal development and synaptic function.”

“Comparison of protein features across exon types To investigate protein features associated with each class of AS exon, we first mapped AS exons to annotated proteomes by translating each alternative exon and its neighboring exons in all 6 frames, and aligning them to the canonical UniProt sequence (Consortium., 2014).”

“The assembly of spliceosomes at 50 and 30 splice sites is typically regulated by RNA-binding proteins (RBPs) that recognize proximal cis-elements, referred to as exonic/intronic splicing enhancers and silencers (Chen and Manley, 2009).”

“Previous genetic and functional studies have revealed multiple functions for the Apbb1-Kat5 complex (Cao and Sudhoff, 2001; Stante et al., 2009), and that the loss of Kat5 activity is associated with developmental defects that impact learning and memory (Pirooznia et al., 2012; Wang et al., 2004,…”

“This is in marked contrast to the generally low degree of evolutionary conservation of other types of AS across vertebrate species (Barbosa-Morais et al., 2012; Braunschweig et al., 2014; Merkin et al., 2012).”

“A GO analysis of genes with ASD-associated misregulation of microexons reveals significant enrichment of terms related to axonogenesis and synapse biology (Figure 7E), processes that have been previously implicated in autism (Gilman et al., 2011; Parikshak et al., 2013; Voineagu et al., 2011).”

“Disorder was predicted using IUPred (Dosztányi et al., 2005) with a disorder cutoff of 0.”

“, 2013) and BioGrid (Chatr-Aryamontri et al., 2013) databases with the dashed lines representing indirect interactions that are either between proteins found within same protein complex but not interacting directly or predicted interactions based on paralogs (Franceschini et al.”

“This is in marked contrast to the generally low degree of evolutionary conservation of other types of AS across vertebrate species (Barbosa-Morais et al., 2012; Braunschweig et al., 2014; Merkin et al., 2012).”

“(Pinto et al., 2014)) whereas partially shaded nodes are genes associated with intellectual disability, FMRP (fragile X mental retardation) or within an ASD CNV but not, as yet, directly implicated in ASD (Banerjee-Basu and Packer, 2010; Pinto et al.”

“(H) Percent of exons with proteins identified as belonging to one or more protein complexes (data from Havugimana et al., 2012).”

“It is also widely established that misregulation of AS plays important roles in altering the growth and invasiveness of various cancers (David and Manley, 2010).”

“Next, to determine the presence of the microexon in relation to the predicted APBB1-PTB1 binding interface, we utilized the CSU algorithm (Sobolev et al., 1999) to analyze the atomic distances between the superimposed APBB1-PTB1 domain and the APP protein fragment.”

“…type- and developmentally-regulated AS events are coordinately controlled by individual RBPs, and that these events are significantly enriched in genes that operate in common biological processes and pathways (Calarco et al., 2011; Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010).”

“of Neuronal Microexons Is”

“To test this hypothesis, we employed luminescence-based mammalian interactome mapping (LUMIER; Barrios-Rodiles et al., 2005; Ellis et al., 2012) and coimmunoprecipitation-western blot assays to investigate whether the insertion of a highly conserved, neural-regulated 6 nt microexon in the nuclear…”

“In addition, we employed previously defined (Venkatesh et al., 2014) clusters of 1-to-1 orthologs for 10 vertebrate species using InParanoid, including”

“A GO analysis of genes with ASD-associated misregulation of microexons reveals significant enrichment of terms related to axonogenesis and synapse biology (Figure 7E), processes that have been previously implicated in autism (Gilman et al., 2011; Parikshak et al., 2013; Voineagu et al., 2011).”

“However, the function of the vast majority of AS events detected to date are not known, and new landscapes of AS regulation remain to be discovered and characterized (Braunschweig et al., 2014; Eom et al., 2013).”

“4), in line with the average length of linear motifs (Davey et al., 2012).”

“(G) Percent of residues overlapping binding motifs predicted by ANCHOR.”

“(G) Percent of residues overlapping binding motifs predicted by ANCHOR.”

“1 (Petersen et al., 2009) and default assignments of accessibility were used.”

“, 2014); PhosphoSite (Hornbeck et al., 2004); Transmembrane predictions, TMHMM (Krogh et al.”

“These data reveal the largest program of neural-regulated AS events defined to date, and that this program is associatedwith a broader range of functional processes and pathways linked to nervous system biology than previously detected (Boutz et al., 2007; Fagnani et al., 2007; Ule et al., 2005).”

“These data reveal the largest program of neural-regulated AS events defined to date, and that this program is associatedwith a broader range of functional processes and pathways linked to nervous system biology than previously detected (Boutz et al., 2007; Fagnani et al., 2007; Ule et al., 2005).”

“…defined, folded domains in proteins, they ell 159, 1511–1523, December 18, 2014 ª2014 Elsevier Inc. 1511 are enriched in regions of protein disorder that typically are surface accessible and embed short linear interaction motifs (Buljan et al., 2012; Ellis et al., 2012; Romero et al., 2006).”

“However, the function of the vast majority of AS events detected to date are not known, and new landscapes of AS regulation remain to be discovered and characterized (Braunschweig et al., 2014; Eom et al., 2013).”

“9 species, for which ENCODE-like data are not available, we performed a de novo search for splice sites using our RNA-Seq dataset (Table S1), as previously described (Curtis et al., 2012).”

“In particular, microexons (Beachy et al., 1985; Coleman et al., 1987), defined here as 3–27 nucleotide (nt)-long exons, have been largely missed by genome annotations and transcriptome profiling studies (Volfovsky et al., 2003; Wu et al., 2013; Wu and Watanabe, 2005).”

“Conversely, unlike longer neural exons (Buljan et al., 2012; Ellis et al., 2012), they are depleted of linear binding motifs (Figures 5G and S5N; p < 0.”

“To further investigate the cell- and tissue-type specificity of microexon regulation, we used RNA-seq data (Sofueva et al., 2013; Zhang et al., 2013, 2014) to compare their inclusion levels in major glial cell types (astrocytes, microglia, and oligodendrocytes), in isolated neurons, and in muscle cells and tissues.”

“, 2013), and Rbfox1 in mouse (Lovci et al., 2013)), or overexpression (nSR100 in human 293T cells (Raj et al.”

“Consistentwith previous results (Fagnani et al., 2007; Pan et al., 2004), there is little overlap (8.5”

“Globular domains known to bind linear motifs and lipids were extracted from the ELM database (Dinkel et al., 2014) and Pfam (Finn et al.”

“The mapping to UniProt was done using EBI SIFTS program (Velankar et al., 2013).”

“, 2010) and alignments of ESTs and cDNAs with genomic sequence (Khare et al., 2012); (2) a ‘‘splice site-based module,’’ utilizing joining of all hypothetically possible EEJ combinations from annotated and de novo splice sites (Han et al.”

“, 2003), processes that have been linked to neurological disorders including ASD (Hussman et al., 2011).”

“that nSR100 promotes neurite outgrowth (Calarco et al., 2009).”

“- AS profiling of splicing factor-deficient or -overexpressing cells: we used available RNA-Seq data from splicing factor knock-downs (RBFOX1 in human neural precursor cells (Fogel et al., 2012), MBNL1 and MBNL2 in human HeLa cells (Han et al.”

“, 2014) was calculated using the cRPKM metric (Labbe et al., 2012).”

“To further investigate the cell- and tissue-type specificity of microexon regulation, we used RNA-seq data (Sofueva et al., 2013; Zhang et al., 2013, 2014) to compare their inclusion levels in major glial cell types (astrocytes, microglia, and oligodendrocytes), in isolated neurons, and in muscle…”

“Apbb1 is an adaptor protein that functions in neurite outgrowth (Cheung et al., 2014; Ikin et al., 2007) and synaptic plasticity (Sabo et al., 2003), processes that have been linked to neurological disorders including ASD (Hussman et al., 2011).”

“…type- and developmentally-regulated AS events are coordinately controlled by individual RBPs, and that these events are significantly enriched in genes that operate in common biological processes and pathways (Calarco et al., 2011; Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010).”

“5 stage) (Li et al., 2013), and Rbfox1 in mouse (Lovci et al.”

“For example, amyotrophic lateral sclerosis-causing mutations in the RBPs TLS/FUS and TDP43 affect AS and other aspects of posttranscriptional regulation (Polymenidou et al., 2012), and changes in the expression of the RBP RBFOX1 have been linked to misregulation of AS in the brains of individuals with autism spectrum…”

“In particular, microexons (Beachy et al., 1985; Coleman et al., 1987), defined here as 3–27 nucleotide (nt)-long exons, have been largely missed by genome annotations and transcriptome profiling studies (Volfovsky et al., 2003; Wu et al., 2013; Wu and Watanabe, 2005).”

“Consistent with this view, recent reports have begun to link individual microexons with neurodevelopmental disorders, including ASD (Zhu et al., 2014), schizophrenia (Ovadia and Shifman, 2011), and epilepsy (Rusconi et al.”

“Most microexons display high inclusion at late stages of neuronal differentiation in genes (e.g., Src [Black, 1991], Bin1, Agrn,Dock9,Shank2, andRobo1) associatedwith axonogenesis and the formation and function of synapses.”

“Taken together with data in Figures 5 and 6, the results suggest that themisregulation ofmicroexons, as well as of longer alternative exons (Corominas et al., 2014; Voineagu et al., 2011), may impact protein-interaction networks that are required for normal neuronal development and synaptic…”

“, 2013), ESRP1 in human PNT2 cells (Dittmar et al., 2012), and Srrm4/nSR100 and Ptbp1 in mouse N2A cells (Raj et al.”

“, 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.”

“Interestingly, mutations in AP1S2 have been previously implicated in phenotypic features associated with ASD and X-linked mental retardation (Borck et al., 2008; Tarpey et al., 2006).”

“In particular, microexons (Beachy et al., 1985; Coleman et al., 1987), defined here as 3–27 nucleotide (nt)-long exons, have been largely missed by genome annotations and transcriptome profiling studies (Volfovsky et al., 2003; Wu et al., 2013; Wu and Watanabe, 2005).”

“…1511–1523, December 18, 2014 ª2014 Elsevier Inc. 1515 (legend continued on next page) 1516 Cell 159, 1511–1523, December 18, 2014 ª2014 Elsevier Inc. formation (Kirchhausen, 2000) and is important for the somatodendritic transport of proteins required for neuronal polarity (Farı́as et al., 2012).”

“These data reveal the largest program of neural-regulated AS events defined to date, and that this program is associatedwith a broader range of functional processes and pathways linked to nervous system biology than previously detected (Boutz et al., 2007; Fagnani et al., 2007; Ule et al., 2005).”

“However, the function of the vast majority of AS events detected to date are not known, and new landscapes of AS regulation remain to be discovered and characterized (Braunschweig et al., 2014; Eom et al., 2013).”

“- Cumulative Distribution plots analyzing nSR100 binding sites: For each upstream intron, the sequence from 3 ́ to 5 ́ starting from the AG dinucleotide (splice site acceptor) was scanned, and the position of the first occurrence of a TGC (a previously-defined nSR100 binding motif (Nakano et al., 2012; Raj et al., 201…”

“In particular, microexons (Beachy et al., 1985; Coleman et al., 1987), defined here as 3–27 nucleotide (nt)-long exons, have been largely missed by genome annotations and transcriptome profiling studies (Volfovsky et al., 2003; Wu et al., 2013; Wu and Watanabe, 2005).”

“…studies have revealed multiple functions for the Apbb1-Kat5 complex (Cao and Sudhoff, 2001; Stante et al., 2009), and that the loss of Kat5 activity is associated with developmental defects that impact learning and memory (Pirooznia et al., 2012; Wang et al., 2004, 2009) (see Discussion).”

“, 2014), schizophrenia (Ovadia and Shifman, 2011), and epilepsy (Rusconi et al.”

“AS events located in these regions are predicted to participate in interactions with proteins and other ligands (Buljan et al., 2012; Weatheritt et al., 2012).”

“…type- and developmentally-regulated AS events are coordinately controlled by individual RBPs, and that these events are significantly enriched in genes that operate in common biological processes and pathways (Calarco et al., 2011; Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010).”

“Switch-like Regulation of Microexons during Neuronal Differentiation (A) Heatmap of PSI changes (DPSIs) between time points during differentiation of ESCs to glutamatergic neurons in vitro (Hubbard et al., 2013).”

“…studies have revealed multiple functions for the Apbb1-Kat5 complex (Cao and Sudhoff, 2001; Stante et al., 2009), and that the loss of Kat5 activity is associated with developmental defects that impact learning and memory (Pirooznia et al., 2012; Wang et al., 2004, 2009) (see Discussion).”

“, 2014), schizophrenia (Ovadia and Shifman, 2011), and epilepsy (Rusconi et al., 2014).”

“Evolutionary analysis of neural-regulated cassette exons We analyzed three different aspects of evolutionary conservation of neural-regulated alternative exons (Irimia et al., 2009).”

“Previous genetic and functional studies have revealed multiple functions for the Apbb1-Kat5 complex (Cao and Sudhoff, 2001; Stante et al., 2009), and that the loss of Kat5 activity is associated with developmental defects that impact learning and memory (Pirooznia et al., 2012; Wang et al., 2004,…”

“Interestingly, mutations in AP1S2 have been previously implicated in phenotypic features associated with ASD and X-linked mental retardation (Borck et al., 2008; Tarpey et al., 2006).”

“Apbb1 is an adaptor protein that functions in neurite outgrowth (Cheung et al., 2014; Ikin et al., 2007) and synaptic plasticity (Sabo et al.”

“Apbb1 is an adaptor protein that functions in neurite outgrowth (Cheung et al., 2014; Ikin et al., 2007) and synaptic plasticity (Sabo et al.”

“Supporting such functions, an alternative microexon overlapping the SH3A domain of Intersectin 1 (Itsn1) has been reported to promote an interaction with Dynamin 1 and was proposed to modulate roles of Itsn1 in endocytosis, cell signaling, and/or actin-cytoskeleton dynamics (Dergai et al., 2010).”

“A neural-specific microexon in Protrudin/Zfyve27 was recently shown to increase its interaction with the vesicle-associated membrane protein-associated protein (VAP) and to promote neurite outgrowth (Ohnishi et al., 2014).”

“…studies have revealed multiple functions for the Apbb1-Kat5 complex (Cao and Sudhoff, 2001; Stante et al., 2009), and that the loss of Kat5 activity is associated with developmental defects that impact learning and memory (Pirooznia et al., 2012; Wang et al., 2004, 2009) (see Discussion).”

“We have also completed an effort to capture all recently described unannotated microexons (18) into GENCODE, and further added an additional 146 novel microexons mined from public SLRseq data (19).”

“…Arises from Weak Cryptic Splice Variants Alternative splicing is a major mode of gene regulation that serves to increase the diversity of transcripts in different tissues and developmental stages, and its dysregulation is associated with disease processes (Irimia et al., 2014; Wang et al., 2008).”

“Finally, several studies have demonstrated that genes carrying microexons are preferentially expressed in the brain and their splicing is dysregulated in ASD (30, 82, 83).”