提前终止密码子的细胞核内识别和滞留机制研究

Nonsense-mediated mRNA decay (NMD) is a specific surveillance mechanism, which detects and rapidly degrades mRNAs containing premature termination codons (PTCs). Eliminating PTC-containing mRNAs (PTC+ mRNAs) is vital because these mRNAs encode C-terminally truncated proteins, which may possess dominant-negative or deleterious gain-of-function activity. However，to date, it remains unclear whether PTC recognition and PTC-triggered mRNA degradation take place exclusively in the cytoplasm, in both the nucleus and cytoplasm, and/or during mRNA export. Combining microinjection with fluorescence in situ hybridization, we unexpectedly found that mRNAs containing any type of PTC (UAG, UGA, UAA), are detained in the nucleus whereas their wild type counterparts rapidly accumulate in the cytoplasm in a reading-frame dependent manner. Detaining PTC+ mRNA is not due to defects or altered kinetics of RNA processing. Next, we plan to investigate the mechanism for recognition and nuclear detention of PTC+ mRNAs. This work will not only advance our knowledge of cellular mRNA surveillance mechanism, but also provide insights into therapies for PTC-related diseases.

无义密码子介导的mRNA降解（NMD）是一种识别并快速降解含有提前终止密码子(PTC) mRNA的质量监控机制。NMD对于细胞正常生命活动至关重要，因为含PTC的mRNA可能翻译产生截短形式的蛋白，从而导致细胞功能异常。然而，至今为止，人们仍然不清楚含PTC的mRNA细胞内的识别位点。通过显微微注射和荧光原位杂交实验，我们发现含有PTC的mRNA被延迟滞留在细胞核内。初步实验结果表明这些 mRNA的滞留并非由于pre-mRNA的核内加工异常导致，而是严格依赖于读码框的完整性。在本项目中，我们将深入探索含PTC的mRNA的识别和核内滞留的机制与功能。本项目不仅将加深我们对细胞mRNA质量监控机制的理解，还将为PTC引发疾病的治疗提供新的思路。

在基因表达过程中存在很多质检步骤来保证基因表达的高保真性。提前终止密码子(PTC)介导的mRNA降解(NMD)是一种转录后水平的监控机制，可以检测并迅速降解含PTC的mRNA(PTC+mRNA)。然而，关于PTC在细胞内的识别位点存在广泛争议，至今还不清楚。在本项目中，我们发现，相对于野生型mRNA，PTC+mRNA延迟滞留在细胞核内，PTC+mRNA的核内加工（剪接和3’加工）没有异常，说明这种核内延迟滞留并非PTC+mRNA的加工缺陷导致。进一步研究表明，PTC引起的核内滞留严格依赖读码框的完整性；我们还排除了PTC+mRNA在细胞质内被检测，并反馈至核内引起PTC+mRNA 的核内滞留的可能。更有意思的是，我们的研究发现PTC+mRNA在细胞核内识别需要起始密码子AUG，帮助核糖体识别翻译起始密码子的位于AUG周边的核糖体识别序列Kozak序列在PTC+mRNA的核内滞留过程中也发挥作用，进一步，我们发现Ribosome和tRNA也参与了PTC+mRNA核内识别。继续研究PTC+mRNA的核内滞留机制，我们发现一个下游的内含子对PTC+mRNA核内滞留发挥功能，我们进一步的实验发现NMD因子UPF1不仅在PTC的核内识别中发挥重要作用并且参与了PTC+mRNA的核内滞留。我们的研究结果可以为细胞提供更早期的mRNA质量监控，这种质量监控机制可能对保证基因表达的高保真性至关重要。