Regulating RNA stability to increase protein production of cells under stress conditions

调节 RNA 稳定性以增加应激条件下细胞的蛋白质产量

• 批准号: 2457522
• 金额: --
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2457522
• 关键词: Regulating; RNA; stability; increase; protein

Survival of cells and organisms in an external environment requires them to reprogramme their cellular pathways to adapt to these stress conditions. During severe stresses such as starvation, cells normally shut down protein synthesis and reduce cell proliferation so as to conserve energy while awaiting the return of normal conditions. In the industrial context, it would be of great economic advantage to be able to grow cells producing medically important proteins under nutrient-poor or nutrient-depleted conditions. We have recently discovered that depletion of the exoribonuclease DIS3L2, in Drosophila and human cells, greatly enhances their growth under starvation conditions and increases protein translation. These results provide the prospect of a novel solution to maximising production of bioactive proteins under nutrient-poor growth conditions. In addition, analysis of the novel stress resistance pathways controlled by DIS3L2 will have implications for understanding DIS3L2-related human diseases such as Wilms' tumour.The aim of this project is to manipulate the expression levels of DIS3L2 in human cell lines to develop a novel cell line which can grow and produce bioactive proteins under nutrient-poor conditions. A range of human cell lines already being used for the production of medically important peptides/proteins (e.g. HEK293 cells) will be screened, firstly by DIS3L2 RNAi and then by CRISPR, to assess their proliferation under nutrient-deficient conditions. Protein production of relevant medically important proteins (e.g. the monoclonal antibody Trastuzumab) from the DIS3L2 mutant cells will also be assessed. To optimise translation in these cells, the student will analyse relevant cellular mechanisms using a range of approaches such as transcriptomic analyses, polysome profiling/ribo-seq, Western blotting and extracellular vesicle trafficking analyses. The project will lead to a new understanding of the cellular response to starvation from the perspective of RNA stability, which has general relevance to cellular metabolism, tissue growth and human disease.

细胞和生物体在外部环境中的生存要求它们重新编程其细胞途径以适应这些应激条件。在饥饿等严重压力下，细胞通常会停止蛋白质合成，减少细胞增殖，以便在等待恢复正常条件的同时保存能量。在工业背景下，能够在营养贫乏或营养枯竭的条件下培养产生医学上重要蛋白质的细胞将具有巨大的经济优势。我们最近发现，果蝇和人类细胞中外切核糖核酸酶Dis3l2的缺失，极大地促进了它们在饥饿条件下的生长，并增加了蛋白质翻译。这些结果为在营养不良的生长条件下最大限度地提高生物活性蛋白的产量提供了一种新的解决方案。此外，对Dis3l2控制的新的抗逆途径的分析将对理解与Dis3l2相关的人类疾病如Wilms瘤具有重要意义。本项目的目的是操纵Dis3l2在人类细胞系中的表达水平，以开发一种新的细胞系，该细胞系能够在营养不良的条件下生长并产生生物活性蛋白。将首先通过Dis3l2 RNAi和CRISPR对一系列已经用于生产具有重要医学意义的多肽/蛋白质的人类细胞株(例如HEK293细胞)进行筛选，以评估它们在营养缺乏条件下的增殖情况。还将评估从Dis3l2突变细胞中产生的相关医学重要蛋白质(例如，单抗曲妥珠单抗)的蛋白质产量。为了优化这些细胞中的翻译，学生将使用一系列的方法来分析相关的细胞机制，例如转录分析、多聚体谱/核糖核酸序列分析、Western blotting和细胞外小泡运输分析。该项目将从RNA稳定性的角度对细胞对饥饿的反应有一个新的理解，RNA稳定性与细胞新陈代谢、组织生长和人类疾病具有普遍相关性。