We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3′-untranslated region (3′-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3′ end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1’s third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

中文翻译：

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Alu 插入介导的 dsRNA 结构形成，预先存在的 Alu 元件是一种致病机制


我们之前在 SPINK1 的 3'-非翻译区 （3'-UTR） 中确定了纯合 Alu 插入变异 （Alu_Ins） 是严重婴儿孤立性胰腺外分泌功能不全的原因。尽管我们确定 Alu_Ins 会导致 SPINK1 mRNA 表达的完全缺失，但确切的机制仍然难以捉摸。在这里，我们旨在通过假设驱动的方法来阐明这些机制。最初，我们推测，由于其特殊位置，Alu_Ins 可以独立破坏 mRNA 3' 末端的形成和/或影响其他转录后过程，例如核输出和翻译。然而，采用 3'-UTR 荧光素酶报告基因测定，发现与野生型相比，Alu_Ins仅导致荧光素酶活性降低 ∼50%，这不足以解释 Alu_Ins 纯合子中的严重胰腺缺陷。然后，我们假设 Alu 元件之间形成的双链 RNA （dsRNA） 结构（调节基因表达的上游机制）可能是原因。使用 RepeatMasker，我们在 SPINK1 的第三个内含子中鉴定了两个 Alu 元件，这两个元件的方向都与 Alu_Ins 相反。通过 RNAfold 预测和全长基因表达测定，我们研究了这些 Alu 重复序列之间的方向依赖性相互作用。我们提供令人信服的证据，将 Alu_Ins 的不利影响与 Alu_Ins 和预先存在的内含子 Alu 序列之间形成的广泛 dsRNA 结构联系起来，包括通过将所有三个 Alu 元件对齐到同一方向来恢复 SPINK1 mRNA 表达。 鉴于 Alu 元件在人类基因组中广泛存在，并且几乎任何基因座都有可能出现新的 Alu 插入，我们的研究结果对于检测和解释疾病基因中的 Alu 插入具有重要意义。