Department of Applied Biochemistry, Institute of Biotechnology, TIB 4/3-2, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.
Biochimie. 2018 Jan;144:169-184. doi: 10.1016/j.biochi.2017.11.003. Epub 2017 Nov 10.
G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. The use of a control composed of samples from all G-quadruplex-forming sequences and their mutated controls ensured that the identified proteins are specific for RNA G-quadruplex structures and are not general RNA-binding proteins. Independent validation experiments based on pull-down assays and Western blotting confirmed the MS data. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). Several of the candidate proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. Interestingly, additional proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions are not only important for the fine-tuning of translation but are also relevant to the regulation of mRNA maturation and may play an important role in tumor biology. Proteomic data are available via ProteomeXchange with identifier PXD005761.
在 mRNA 的 5'UTR 中，G-四链体结构被广泛认为可以抑制翻译而不影响转录。本研究全面分析了结合在癌症相关基因 Bcl-2、NRAS、MMP16 和 ARPC2 的 mRNA 中四个不同 G-四链体基序的蛋白质。在人细胞系 HEK293 中进行蛋白质的代谢标记（稳定同位素标记与细胞培养中的氨基酸，SILAC）后，通过下拉实验富集 G-四链体结合蛋白，并通过 LC-轨道阱质谱进行鉴定。我们发现，在所研究的 G-四链体基序中，存在不同的相互作用模式。虽然 NRAS 和 MMP16 mRNA 中的 G-四链体与少数蛋白质特异性相互作用，但 Bcl-2 和 ARPC2 G-四链体与广泛的蛋白质相互作用伙伴相互作用，分别在至少两个重复中鉴定出 99 和 82 个候选蛋白。使用由所有形成 G-四链体的序列及其突变对照组成的对照样本确保了所鉴定的蛋白质是特定于 RNA G-四链体结构的，而不是一般的 RNA 结合蛋白。基于下拉实验和 Western 印迹的独立验证实验证实了 MS 数据。相互作用伙伴中有许多已知与 RNA 结合的蛋白质，包括多个异质核核糖核蛋白（hnRNPs）。候选蛋白中的几种可能反映了核糖体被 RNA G-四链体结构阻碍。有趣的是，还鉴定出了以前未描述过与 RNA 相互作用的其他蛋白质。候选蛋白的基因本体分析表明，许多相互作用伙伴是已知与肿瘤相关的。大多数鉴定的 RNA G-四链体相互作用蛋白被认为参与转录后过程，特别是剪接。这些发现表明，蛋白-G-四链体相互作用不仅对翻译的微调很重要，而且与 mRNA 成熟的调节有关，可能在肿瘤生物学中发挥重要作用。蛋白质组学数据可通过 ProteomeXchange 获得，标识符为 PXD005761。
