Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.
Department of Biological Chemistry, Center for RNA Biomedicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Nucleic Acids Res. 2022 Sep 23;50(17):9689-9704. doi: 10.1093/nar/gkac739.
SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5'-untranslated region (5'-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5'-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5'-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5'-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5'-UTR structure. However, those mutations that disrupted the helix that anchors the 5'-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5'-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5'-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.
丝氨酸蛋白酶抑制剂 1（SERPINA1）mRNA 编码蛋白酶抑制剂 α-1-抗胰蛋白酶，其转录后水平的调控机制与翻译后机制有关。α-1-抗胰蛋白酶缺乏会导致慢性阻塞性肺疾病（COPD）和肝硬化，5'非翻译区（5'-UTR）的特定变体与 COPD 相关。NM_000295.4 转录本在肺和血液中高表达和翻译，其特征是具有延伸的 5'-UTR，不包含竞争的上游开放阅读框（uORF）。我们发现 NM_000295.4 的 5'-UTR 可以折叠成一个定义明确的多螺旋结构域。我们系统地破坏 NM_000295.4 5'-UTR 中的 mRNA 结构，并测量（通过 SHAPE 定量）RNA 结构和帽依赖性翻译相对于天然序列报告的变化。令人惊讶的是，尽管破坏了局部 RNA 结构，但大多数突变要么没有影响，要么降低了翻译效率。大多数破坏 RNA 结构的突变保留了天然的全局 5'-UTR 结构。然而，那些破坏锚定 5'-UTR 结构域的螺旋的突变产生了三组非天然结构。这三组非天然结构中的两组重新折叠形成了靠近翻译起始位点的稳定螺旋，从而降低了翻译效率。因此，与传统的 5'-UTR 中 RNA 结构主要抑制翻译的模型相反，NM_000295.4 5'-UTR 的复杂折叠通过促进翻译起始位点的可及性，创造了一种翻译优化的信使。
