Table1_Analysis of Pathogenic Pseudoexons Reveals Novel Mechanisms Driving Cryptic Splicing.XLSX

Understanding pre-mRNA splicing is crucial to accurately diagnosing and treating genetic diseases. However, mutations that alter splicing can exert highly diverse effects. Of all the known types of splicing mutations, perhaps the rarest and most difficult to predict are those that activate pseudoexons, sometimes also called cryptic exons. Unlike other splicing mutations that either destroy or redirect existing splice events, pseudoexon mutations appear to create entirely new exons within introns. Since exon definition in vertebrates requires coordinated arrangements of numerous RNA motifs, one might expect that pseudoexons would only arise when rearrangements of intronic DNA create novel exons by chance. Surprisingly, although such mutations do occur, a far more common cause of pseudoexons is deep-intronic single nucleotide variants, raising the question of why these latent exon-like tracts near the mutation sites have not already been purged from the genome by the evolutionary advantage of more efficient splicing. Possible answers may lie in deep intronic splicing processes such as recursive splicing or poison exon splicing. Because these processes utilize intronic motifs that benignly engage with the spliceosome, the regions involved may be more susceptible to exonization than other intronic regions would be. We speculated that a comprehensive study of reported pseudoexons might detect alignments with known deep intronic splice sites and could also permit the characterisation of novel pseudoexon categories. In this report, we present and analyse a catalogue of over 400 published pseudoexon splice events. In addition to confirming prior observations of the most common pseudoexon mutation types, the size of this catalogue also enabled us to suggest new categories for some of the rarer types of pseudoexon mutation. By comparing our catalogue against published datasets of non-canonical splice events, we also found that 15.7% of pseudoexons exhibit some splicing activity at one or both of their splice sites in non-mutant cells. Importantly, this included seven examples of experimentally confirmed recursive splice sites, confirming for the first time a long-suspected link between these two splicing phenomena. These findings have the potential to improve the fidelity of genetic diagnostics and reveal new targets for splice-modulating therapies.

阐明前体mRNA（pre-mRNA）剪接的分子机制，对于精准诊断与治疗遗传病至关重要。然而，改变剪接模式的突变可引发高度多样的生物学效应。在所有已知的剪接突变类型中，激活伪外显子（pseudoexon）的突变或许是最罕见、最难预测的一类，这类突变有时也被称为隐蔽外显子。与其他要么破坏现有剪接事件、要么重定向剪接通路的剪接突变不同，伪外显子突变似乎会在内含子区域内催生全新的外显子。由于脊椎动物的外显子定义需要众多RNA基序的协同排布，人们或许会认为，仅当内含子DNA发生重排并偶然创造出新外显子时，才会出现伪外显子。令人意外的是，尽管这类重排突变确实存在，但伪外显子更常见的诱发因素是深层内含子单核苷酸变异，这不禁引发一个关键问题：为何这些位于突变位点附近、具备类外显子特征的潜在序列，未因更高效剪接带来的进化选择优势而从基因组中被清除？相关答案或许藏于递归剪接、毒性外显子剪接等深层内含子剪接过程之中。由于这类过程利用的内含子基序可与剪接体发生无害结合，相关区域可能比其他内含子区域更易发生外显子化事件。我们曾推测，对已报道的伪外显子开展全面研究，不仅能检测其与已知深层内含子剪接位点的对齐关联，还可实现对新型伪外显子类别的系统表征。在本研究报告中，我们呈现并分析了一份涵盖逾400个已发表伪外显子剪接事件的汇编目录。除验证了此前关于最常见伪外显子突变类型的观测结果外，该目录的规模还让我们得以为部分罕见伪外显子突变类型提出全新的分类标准。通过将该目录与已发表的非经典剪接事件数据集进行比对分析，我们还发现，15.7%的伪外显子在非突变细胞的一个或两个剪接位点处表现出一定的剪接活性。值得注意的是，其中包含7个经实验证实的递归剪接位点案例，首次验证了这两种剪接现象之间长期以来被学界推测存在的关联。上述研究结果有望提升遗传诊断的精准性，并为剪接调控疗法发掘全新的靶点。