second, as events in hescs were more similar, whereas the as events in derived nps were consistent with or intermediate to the benchmark hcns - scns, likely reflecting differences in the cell lines and / or differentiation protocols. in addition, we tested the as patterns of reap [ + ] exons from ehbp1, slk, and rai14 in a panel of differentiated human tissues ( figure 7c ). the reap [ + ] alternative exon in the rai14 ( retinoic acid induced 14 ) gene was observed to have the same as pattern in nps as in frontal and temporal cortex and in several other, non - brain adult tissues, such as heart and spleen. the as pattern of the reap [ + ] exon in the slk gene in nps was similar to most differentiated tissues; however, the relatively strong inclusion of the exon in hescs was unique. even in esophagus, kidney, liver, and prostate, both isoforms were present. the relative ratio of the exon - included to exon - skipped isoforms in slk likely represents an esc - specific as signature. the alternative exon in the ehbp1 gene was unusual. the exon was included in hescs but also in frontal cortex and temporal cortex, a finding that was unexpected given the exclusion of the exon in hcns - scns ( figure 7c ). the as pattern in hcns - scns may represent a transient, early neuronal molecular change. functional and expression characteristics of reap [ + ] genesin total, 1, 500 genes were identified that contained 1, 737 reap [ + ] exons, 68 % of which lacked prior transcript ( est / cdna ) evidence for as. to determine whether genes that contained reap [ + ] exons, which we refer to as reap [ + ] genes, are biased toward particular biological activities, reap [ + ] genes were compared to a set of reap analyzed genes not found to have reap [ + ] exons ( reap [ − ] genes ). a gene ontology analysis revealed that reap [ + ] genes are enriched for go molecular function categories “ atp binding, ” “ helicase activity, ” “ protein serine / theronine kinase activity, ” “ small gtpase regulatory / interacting protein activity, ” and “ thyroid hormone receptor binding ” ( table 1 ). in terms of go biological process categories, reap [ + ] genes were more frequently involved in “ ubiquitin cycle. ” similar results were obtained when we compared reap [ + ] genes to all human genes that did not contain reap [ + ] exons ( table 1 ) [ 55 ]. table 1significantly enriched gene ontology terms in reap [ + ] genes ( cutoff of two significant “ outliers ” per probeset ) next we asked if reap [ + ] genes are differentially expressed in hescs compared to nps and vice versa. for this analysis, the t - statistics computed above measuring the enrichment of a gene in hescs relative to nps was utilized for only reap - analyzed genes. at a defined absolute - valued cutoff, genes were divided into three categories: “ enriched in hescs, ” “ enriched in np, ” or “ unchanged ” ( figure 8a ). increasing the t - statistic cutoff from one to five, the fraction of reap [ + ] genes relative to reap - analyzed genes remained constant in the “ unchanged ” categories ( figure 8b ). however, the fraction of reap [ + ] exons decreased significantly in “ enriched in hescs ” and “ enriched in nps ” categories. if we increased the cutoffs on genes that were randomly assigned as reap [ + ] and reap [ − ], controlling for the same number of genes in each category, we observed that the fraction of reap [ + ] exons remained unchanged for all three categories ( figure 8c ). to illustrate, at a cutoff of five, 10 % ( 29 of 267 ) of enriched np genes were reap [ + ] genes and 8. 8 % ( 102 of 1, 162 ) of enriched hesc genes were reap [ + ], significantly different ( p < 0. 000005 ) from the random control where ∼14 % of enriched np and enriched hesc genes were reap [ + ]. at a cutoff of five, 14 % ( 1, 368 of 9, 636 ) of genes that were expressed at similar levels between hescs and nps were reap [ + ]. our results suggested that a strategy of focusing on differentially expressed genes would miss at least 14 % of transcriptionally unchanged genes that may nevertheless have functional as differences between hescs and nps. figure 8analysis of reap [ + ] genes relative to transcriptional differences ( a ) histogam of t - statistics computed from gene - level signal estimates measuring the enrichment of genes in hesc and in np. genes on the right of the vertical line at 5 were designated enriched in hesc and genes on the left of the vertical line at −5 were designated enriched in np; genes in between −5 and 5 were designated as “ unchanged ” or expressed similarly in hesc and np. ( b ) vertical bars representing the percentage of reap [ + ] genes out of all genes in the different classifications ( dashed bar: “ enriched in hesc ”; black filled bar: “ unchanged ”; white filled bar: “ enriched in np ” ), at different cutoffs of 1 to 5. ( c ) set of genes where reap [ + ] designation was randomly chosen. similar representation as in ( b ). conserved intronic splicing regulatory elements proximal to reap [ + ] hesc and np exonsmany, if not most, alternative exons undergo cell type – specific regulation by the binding of trans - factors to splicing regulatory cis - elements located proximal to or within the exons. as many tissue - specific splicing cis - regulatory elements were localized in intronic regions of as exons, we focused on the identification of intronic splicing regulatory elements ( isres ) proximal to reap [ + ] exons. in addition, we wanted to identify both common and cell type – specific isres. three sets of exons were generated: ( i ) reap [ + ] exons that were predicted to be included in nps and skipped in hescs ( reap [ + ] np ); ( ii ) reap [ + ] exons that were predicted to be included in hescs and skipped in nps ( reap [ + ] hesc ); and ( iii ) all reap [ − ] exons. regions of 400 base pairs flanking the exons were targeted for search. initially, 5 - mers that were significantly enriched between the upstream and downstream intronic regions of reap [ + ] np and reap [ + ] es relative to reap [ − ] exons were enumerated. we were not able to identify 5 - mers that were statistically significantly different. next, we focused on splicing signals that were conserved across mammalian genomes as a way of enhancing the signal of detecting functional splicing regulatory sequences [ 66 ]. exons that were orthologous across human, dog, rat, and mouse were obtained and the flanking intronic regions were aligned ( 400 bases upstream and downstream separately; figure 9a ). we enumerated k - mers that were perfectly conserved across all four genomes in the upstream ( and downstream ) intronic regions. each conserved k - mer was attributed a χ score representing its enrichment in a set of exons relative to another set of exons. the higher the score, the more frequent the conserved k - mer was in the first set relative to the second set. as a negative control, the associations between reap scores and exons were shuffled. the enrichment scores for all downstream intronic 5 - mers for shuffled reap [ + ] np versus set reap [ − ] exons ( x - axis ), and for shuffled reap [ + ] es exons versus reap [ − ] exons ( y - axis ) were displayed ( figure 9b ). at a χ cutoff of three, which corresponded to a p - value of 0. 0015, the majority of 5 - mers were not significantly enriched in either shuffled set. confident that no association of k - mers with shuffled reap exons were found; we repeated the analyses for upstream and downstream intronic 5 - mers for the original unshuffled sets. we identified 68 conserved 5 - mers enriched upstream of reap [ + ] np exons; and 34 5 - mers enriched upstream of reap [ + ] es exons ( figure 9c; table s4 ). of the 5 - mers that were significantly enriched upstream of reap [ + ] np exons, we identified a u - rich motif ( uuuuu ), a gu - rich motif ( gugug ), and a cu - rich motif ( ccucu, cucuc, ucucu, gcucu ).