To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem–loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

为了阐明外显子序列在人类RNA转录物剪接中的程度和作用，我们在三外显子小基因中进行了51 nt内部外显子的饱和诱变。调查了所有可能的单核苷酸和串联二核苷酸取代。使用高通量遗传学，测定了5560个小基因分子在人HEK293细胞中的剪接。多达70％的突变产生剪接增加或减少的实质性（大于两倍）表型。在所有预测的二级结构元素中，只有一个15nt的茎-环显示出与剪接的强相关性，且呈负相关。突变分子和与剪接体形成有关的蛋白（U2AF35，U2AF65，U1A和U1-70K）之间外显子蛋白复合物的体外形成与剪接效率相关，建议将外显子定义为受大多数突变影响的步骤。发现在CISBP-RNA数据库中报告的数十种人类RNA结合蛋白结构域的相对结合亲和力与剪接效率呈正相关或负相关，超过了同时适合51-nt测试外显子的相关性。这些功能性蛋白质结合相关性的大量指向动态且异质的前mRNA分子群体，每个分子都对结合蛋白的特定集合做出反应。