【专业英语- Pre-mRNA alternative splicing/O-GlcNAc】

Pre-mRNA alternative splicing is a major source of proteomic and functional diversity . In humans, ~95% of multi-exon genes are subjected to alternative splicing , and splicing defects are associated with a variety of genetic diseases . Common alternative splicing mechanisms include exon skipping, intron retention, alternative 5′ or 3′ splice site usage, and mutually exclusive splicing. Mutually exclusive splicing occurs when only one exon from a cluster of variable exons is spliced into a specific mRNA product . The most astonishing example of this can be found in the Drosophila melanogaster Down’s syndrome cell adhesion molecule 1 (Dscam1) gene, which potentially generates 38,016 different isoforms through mutually exclusive splicing of exon clusters 4, 6, 9, and 17 . Growing evidence has revealed that the notable diversity of Dscam1 isoforms is required for both neuronal wiring and immune defense .

前mRNA替代剪接是蛋白质组学和功能多样性的主要来源。在人类中，~95%的多外显子基因受到交替剪接，剪接缺陷与多种遗传疾病有关。常见的替代剪接机制包括外显子跳跃、内含子保留、替代 5' 或 3' 剪接位点使用以及互斥剪接。当可变外显子簇中只有一个外显子被拼接到特定的mRNA产物中时，就会发生互斥剪接。最令人惊讶是，可以在黑腹果蝇唐氏综合征细胞粘附分子 1 （Dscam1） 基因中找到，该基因通过外显子簇 4、6、9 和 17 的相互排斥剪接可能产生 38，016 种不同的亚型。越来越多的证据表明，Dscam1亚型的显着多样性是神经元布线和免疫防御所必需的。

O-linked N-acetylglucosamine (O-GlcNAc) is a prevalent protein modification that plays fundamental roles in both cell physiology and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase (OGT). The study of protein O-GlcNAc function is limited by the lack of tools to control OGT activity with spatiotemporal resolution in cells. Here, we report light control of OGT activity in cells by replacing a catalytically essential lysine residue with a genetically encoded photocaged lysine. This enables the expression of a transiently inactivated form of OGT, which can be rapidly reactivated by photo-decaging. We demonstrate the activation of OGT activity by monitoring the time-dependent increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based quantitative proteomics. We further apply this activation strategy to control the morphological contraction of fibroblasts. Furthermore, we achieved spatial activation of OGT activity predominantly in the cytosol. Thus, our approach provides a valuable chemical tool to control cellular O-GlcNAc with much needed spatiotemporal precision, which aids in a better understanding of O-GlcNAc function.

O-连接的N-乙酰葡糖胺（O-GlcNAc）是一种普遍的蛋白质修饰，在细胞生理学和病理学中都起着重要作用。O-GlcNAc仅由O-乙酰葡糖胺转移酶（OGT）催化。蛋白质O-乙酰葡糖胺功能的研究受到缺乏控制细胞中时空分辨率的OGT活性的工具的限制。在这里，我们报告了通过用基因编码的光笼赖氨酸替换催化必需赖氨酸残基来控制细胞中OGT活性的光控制。这使得短暂失活的OGT的表达成为可能，其可以通过光降解快速重新激活。我们通过监测细胞O-乙酰葡糖胺对时间依赖性延长和使用基于质谱的定量蛋白质组学分析糖蛋白来证明OGT活性的激活。我们进一步应用这种激活策略来控制成纤维细胞的形态收缩。此外，我们实现了主要在细胞质中OGT活性的空间激活。因此，我们的方法提供了一种有价值的化学工具来控制细胞O-乙酰葡糖胺，具有急需的时空精度，这有助于对O-乙酰葡糖胺功能更好的了解。