Krogh Nicolai, Pietschmann Max, Schmid Manfred, Jensen Torben Heick, Nielsen Henrik
Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
RNA. 2017 May;23(5):683-695. doi: 10.1261/rna.059337.116. Epub 2017 Feb 3.
The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete to replace the mRNA mG cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2', 5' phosphodiester bond. We show that the ribozyme functions in vivo in the budding yeast presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the mG cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its mG-capped counterpart, consistent with a key role for the mG cap in mRNA turnover. Our study emphasizes important activities of the mG cap and suggests new utilities of lariat capping as a molecular tool in vivo.
真核生物mRNA的5'帽结构对其加工、运输、翻译和稳定性至关重要。帽的多种功能以及几乎所有mRNA都携带相同类型的帽这一事实，使得在基因表达的各个步骤中体内分析帽功能变得困难。我们使用了来自黏菌的套索帽状核酶（LCrz），用一个微小的套索取代单个报告mRNA物种的mRNA mG帽，其中第一个和第三个核苷酸通过2',5'磷酸二酯键连接。我们表明，该核酶在芽殖酵母体内发挥作用，可能无需辅因子，并且套索帽化在转录过程中同时发生。套索帽化的报告mRNA被有效地输出到细胞质中，在那里发现它被寡腺苷酸化并均匀分布。寡腺苷酸化形式和带有模板化多聚（A）尾的套索帽化mRNA翻译效率都很低，这突出了mG帽在翻译中的关键重要性。最后，与mG帽化的对应物相比，套索帽化的RNA半衰期长三倍，这与mG帽在mRNA周转中的关键作用一致。我们的研究强调了mG帽的重要活性，并提出了套索帽化作为体内分子工具的新用途。
