Effect of SRP54 depletion on mRNA expression in cultured human cells

Regulation of Aberrant Protein Production (RAPP) is a recently discovered protein quality control pathway that monitors proteins during their synthesis at the ribosome and degrades their mRNA if the proteins are not able to interact with their natural partners. In this study we induced RAPP by knockdown of SRP54, a Signal Recognition Particle subunit, and studied its effect at the full transcriptome level. The mRNA expression levels of many secretory and membrane proteins were decreased demonstrating the generality of the pathway. The RAPP activation leads to dramatic upregulation of the specific network of ribosome-associated HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. The results demonstrate a complex nature of mRNA and protein quality control at the ribosome. Two experimental condition (treated and control cells). Treatment: HeLa Tet-On cells were transfected with siRNA for SRP54 to knockdown SRP54. Control: HeLa Tet-On cells were transfected with control siRNA. Each experimental condition included three independent biological replicates.

异常蛋白质生成调控（Regulation of Aberrant Protein Production, RAPP）是近年来新发现的蛋白质质量控制通路，可在蛋白质于核糖体合成过程中实施监控，若蛋白质无法与其天然结合伴侣相互作用，则降解其对应的信使RNA（mRNA）。本研究通过敲低信号识别颗粒（Signal Recognition Particle, SRP）亚基SRP54来诱导RAPP通路激活，并在全转录组层面探究其调控效应。众多分泌蛋白与膜蛋白的mRNA表达水平出现下调，证实了该通路的普适性。RAPP通路激活可显著上调核糖体结合型HSP70/40/90分子伴侣网络（HSPA1A、DNAJB1、HSP90AA1等）的表达，提升核糖体结合泛素化水平，并改变RPS27与RPS27L的表达水平，提示存在核糖体重排现象。研究结果揭示了核糖体层面mRNA与蛋白质质量控制的复杂机制。本研究设置两组实验条件：处理组与对照组细胞。处理组：将HeLa Tet-On细胞转染靶向SRP54的小干扰RNA（siRNA）以敲低SRP54表达；对照组：将HeLa Tet-On细胞转染阴性对照siRNA。每组实验条件均设置3次独立生物学重复。