it is known that the heterogeneous ribonucleoprotein c ( hnrnp c ) binding site obtained by selex is five “ u ” s [ 67 ]. gu - rich sequences in flanking intronic regions were shown to bind to splicing factor etr - 3 to regulate as [ 68 ]. cu - rich sequences were shown to bind the splicing factor ptb [ 69 ]. of the 5 - mers enriched upstream of reap [ + ] es exons, we observed cuaac, which resembled the splicing branch - signal. of the six 5 - mers that were enriched upstream of both reap [ + ] np and reap [ + ] es exons, we identified gcaug, which was previously shown to be an intronic splicing cis - element for the mammalian fibronectin and calcitonin / cgrp genes [ 70 – 72 ]. more recently, both mammalian fox1 and 2 have been demonstrated to regulate alternatively spliced exons via ugcaug binding sites in neighboring introns in neuronal cell cultures [ 73 ]. figure 9conserved intronic cis - elements enriched proximal to reap [ + ] alternative exons ( a ) schematic describing the enumeration of intronic elements across 400 bases of flanking mammalian introns ( human, dog, rat, and mouse ). red and green horizontal bars represent conserved intronic elements and nonconserved elements, respectively. internal exons were divided into reap [ + ] np, reap [ + ] es, and reap [ − ] exons. the χ statistic was computed to represent the enrichment of conserved elements in intronic regions flanking reap [ + ] np versus reap [ − ] exons ( x - axis ), and reap [ + ] es versus reap [ − ] exons ( y - axis ). the sign represented the direction of change, i. e., positive if enriched in introns flanking reap [ + ] versus reap [ − ] exon. each conserved 5 - mer was associated with two numbers: the enrichment in introns proximal to reap [ + ] np versus reap [ − ] exons ( x - axis ), and reap [ + ] es versus reap [ − ] exons ( y - axis ). ( b ) downstream intronic regions, where the association between reap [ + ] designation and the exons was shuffled. ( c ) upstream intronic regions. circled 5 - mers in the upper right quadrant represent conserved 5 - mers enriched in the upstream intronic regions of reap [ + ] np and reap [ + ] es exons. ( d ) downstream intronic regions. circled 5 - mers in the upper right quadrant represent conserved 5 - mers enriched in the downstream intronic regions of reap [ + ] np and reap [ + ] es exons. eighteen conserved 5 - mers were significantly enriched in the downstream introns of reap [ + ] es exons; and 76 5 - mers were enriched downstream of reap [ + ] np exons ( table s4, figure 9d ). we identified a motif cucau resembling the nova binding site ycay [ 74 ], and a g - rich motif ( agggg, gggga, ggggc, ggggg, ggggu ) enriched in the introns downstream of reap [ + ] es exons. g - rich motifs had previously been shown to be part of a bipartite signal that silences as exons [ 75 ]. of the five 5 - mers that were enriched downstream of both reap [ + ] np and reap [ + ] es exons, gcaug and a u - rich motif ( uuuuu ) were identified. we concluded that potential isres were enriched proximal to a subset of reap [ + ] exons; in particular, the fox1 / 2 binding site gcuag may play a regulatory role in controlling as events in hescs and nps. the ability of escs to generate all three embryonic germ layers has raised the exciting possibility that hescs may become an unlimited source of cells for transplantation therapies involving organs or tissues such as the liver, pancreas, blood, and nervous system, and become tools to explore the molecular mechanisms of human development. despite such interests, relatively little is understood about the molecular mechanisms defining their pluripotency and the molecular changes important for hescs to differentiate into specific cell types. to understand these events, protocols are still being developed to differentiate escs into a variety of lineages. of particular biomedical interest is in the capacity of hescs to be differentiated into a self - renewing population of nps that can be then further coaxed into a variety of neuronal subtypes, such as dopaminergic neurons that are important in the treatment of parkinson disease or cholinergic neurons for als ( amyot rophic lateral sclerosis ). while many microarray studies have explored molecular differences between hescs and derived nps, most, if not all, have focused on transcriptional changes. these studies have largely ignored intermediate rna processing events prior to and during translation. in recent years, as has gained momentum as being important in development, apoptosis, and cancer. reap, a regression - based method for analyzing exon array data was introduced, and was applied to discover as events in hescs, their derived nps, and in hcns - scns. reap was based on the assumptions that most exons in the gene of interest and in the genome are constitutively spliced and that outliers in a linear pairwise comparison of the signal estimates for probesets in a gene could be detected using a robust regression - based approach. reap predictions were found to correlate well with transcript - based methods for identifying alternative exons, which interestingly suggested that current databases of transcript information, albeit not specifically enriched for hesc or nps, in aggregate are nevertheless predictive of as events in hesc and np. in addition, reap [ + ] exons were also enriched for acescan - predicted evolutionarily conserved exons [ 55 ]. as acescan utilized a different set of information from reap, the agreement between both algorithms served to further validate the predicted alternative exons. additional studies in mouse escs and neural derivatives will be necessary to determine if these as events are indeed preserved in these analogous and orthologous cell types. our finding that only a minority of as events was common between various hesc to np comparisons is intriguing. a possible explanation is that the cell lines were not only genetically different, but were also exposed to different isolation and culture conditions. in addition, the different differentiation protocols established as optimal for generating nestin and sox1 positive neural precursors may lead to vastly different molecular changes. it is likely that post - transcriptional changes such as as may be more variable despite the cells being at acknowledged “ end - points ” defined by a limited set of immunoh istochemical markers. our results are consistent with a recent study that showed that while two well - established hesc lines differentiate into functional neurons, the two lines exhibited distinc t differentiation potentials, suggesting that some preprogramming had occurred [ 76 ]. in particular, microrna profiling revealed significant expression differences between the two hesc lines, suggesting that micrornas, known post - transcriptional regulators, may sway the differentiation properties of the cell lines [ 76 ]. we postulated that as events may serve also to bias the differentiation spectrum of the cells, an important avenue for future work. experimental validation of reap [ + ] exons suggested a high specificity at the expense of relatively moderate sensitivity. we believe that the high fp rates may arise from cross - hybri diz ation effects that remained unaccounted for. however, our specificity of 77 % at the cutoff of two significant outliers per probeset allowed us to estimate that at least 1, 336 of 1, 737 reap [ + ] exons were true as events that changed during neuronal differentiation of hesc cells, and / or were different between endogeneous nps and hesc. on average, 7 % of all human exons have been estimated by transcript data to undergo as; thus reap ' s validation rate of 60 % at the cutoff of two is 73 - fold ( 60 / 7 ) higher than expected. in addition, we validated nine novel as events that disting uish hescs and nps. consistent with our computational results, we observed that the as patterns in hcns - scns were not always similar to those of the derived nps.