Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Open Biol. 2020 Nov;10(11):200313. doi: 10.1098/rsob.200313. Epub 2020 Nov 25.
The m7G cap marks the 5' end of all eukaryotic mRNAs, but there are also capped ends that map downstream within spliced exons. A portion of the mRNA transcriptome undergoes a cyclical process of decapping and recapping, termed cap homeostasis, which impacts the translation and stability of these mRNAs. Blocking cytoplasmic capping results in the appearance of uncapped 5' ends at native cap sites but also near downstream cap sites. If translation initiates at these sites the products would lack the expected N-terminal sequences, raising the possibility of a link between mRNA recapping and proteome complexity. We performed a shotgun proteomics analysis on cells carrying an inducible inhibitor of cytoplasmic capping. A total of 21 875 tryptic peptides corresponding to 3565 proteins were identified in induced and uninduced cells. Of these, only 29 proteins significantly increased, and 28 proteins significantly decreased, when cytoplasmic capping was inhibited, indicating mRNA recapping has little overall impact on protein expression. In addition, overall peptide coverage per protein did not change significantly when cytoplasmic capping was inhibited. Together with previous work, our findings indicate cap homeostasis functions primarily in gating mRNAs between translating and non-translating states, and not as a source of proteome complexity.
m7G 帽结构标记了所有真核生物 mRNA 的 5' 端，但也存在映射到拼接外显子下游的帽端。一部分 mRNA 转录本经历一个称为帽状结构动态平衡的循环去帽和再帽过程，该过程会影响这些 mRNA 的翻译和稳定性。阻断细胞质加帽会导致在天然帽位点和附近下游帽位点出现无帽 5' 端。如果在这些位点起始翻译，产物将缺乏预期的 N 端序列，这就提出了 mRNA 再帽和蛋白质组复杂性之间可能存在联系。我们对携带诱导型细胞质加帽抑制剂的细胞进行了鸟枪法蛋白质组学分析。在诱导和未诱导细胞中鉴定到了总共 21875 个对应于 3565 种蛋白质的胰蛋白酶肽。其中，当细胞质加帽被抑制时，只有 29 种蛋白质显著增加，28 种蛋白质显著减少，这表明 mRNA 再帽对蛋白质表达的总体影响很小。此外，当细胞质加帽被抑制时，每个蛋白质的总肽覆盖率没有显著变化。结合以前的工作，我们的发现表明帽状结构动态平衡主要在将 mRNA 从翻译状态和非翻译状态之间进行门控，而不是作为蛋白质组复杂性的来源。
