Elsevier

Methods

Volume 167, 1 September 2019, Pages 124-133
Methods

Probes and drugs that interfere with protein translation via targeting to the RNAs or RNA-protein interactions

https://doi.org/10.1016/j.ymeth.2019.06.004Get rights and content

Highlights

  • The general process and mechanism of protein synthesis is introduced.

  • The finding of chemical probes targeting proteins involved in this process is discussed.

  • The therapeutic potential of these probes is also discussed to give a comprehensive understanding.

Abstract

Protein synthesis is critical to cell survival and translation regulation is essential to post-transcriptional gene expression regulation. Disorders of this process, particularly through RNA-binding proteins, is associated with the development and progression of a number of diseases, including cancers. However, the molecular mechanisms underlying the initiation of protein synthesis are intricate, making it difficult to find a drug that interferes with this process. Chemical probes are useful in elucidating the structures of RNA-protein complex and molecular mechanism of biological events. Moreover, some of these chemical probes show certain therapeutic benefits and can be further developed as leading compounds. Here, we will briefly review the general process and mechanism of protein synthesis, and emphasis on chemical probes in examples of probing the RNA structural changes and RNA-protein interactions. Moreover, the therapeutic potential of these probes is also discussed to give a comprehensive understanding.

Introduction

All cellular proteins are produced from the mRNA in the process of translation, in which functional ribosomes and protein complexes are involved by sequentially assembling onto the mRNA strands. The orderly and controllable development of various stages of protein synthesis is critical to the growth of normal cells, especially a precise translation initiation. Therefore, it is of great significance to elucidate the molecular mechanism of translation regulation and to search for relevant therapeutic strategies for drug discovery.

Protein translation is a complex process including initiation, extension and termination. Each link depends on the precise interactions between a large number of translation factors and the mRNA or tRNA. Direct structure elucidation will no doubt provides an accurate explanation of the interactions between proteins and RNAs, while this is difficult because of the large number of complex participants. In fact, the study focus in vitro is on structure analysis and binding mode analysis, while the intracellular function study is more difficult. In this case, chemical probes help reveal the molecular mechanism of cellular events because they make them visible or easy to track. In addition, some chemical probes can be further modified to be selective drugs because they can interact specifically with specific targets. Here we aim to review the probes and the related technologies used to study RNA structure changes and RNA-protein interactions during the initiation of translation basing on a brief introduction of the translation process.

Section snippets

General process of eukaryotes translate initiation

During the translation cap-dependent initiation, the elongation-competent 80S ribosome is assembled onto the mRNA chain where the initiator tRNA is base-paired with the initiation codon in the P-site of ribosome [1]. In most of the genes, the 80S complexes are formed by a scanning-dependent manner (Fig. 1). In short, the 43S, 60S and 80S complexes are formed successively, in which the 43S complex is responsible for scanning the 5′-untranslated regions (5′-UTR) structure to determine the

Cap analogs as probes for mRNA structural and functional studies

The interactions between folded RNA and effector proteins are very common in genome, and are vital for diverse biological functions. Structural elucidation of translation initiation complexes seems to be the most direct way to study the interactions between proteins and mRNAs and the possible mechanism, depending on the studies using mRNA. However, mRNA is a relatively short-lived molecule and usually at the nanomolar or even picomolar level in cells, making it difficult to detect and quantify

RNA labeling methods

The detection of defined RNAs in vivo requires probes with high specificity, sensitivity, and signal-to-background ratio, especially for RNAs with low copy numbers and inside living cells. Thus, instead of using cap analogs to study the structures and binding modes of mRNA with proteins, several protocols for RNA fluorescent and affinity labeling have been developed for in vitro and in vivo studies, including random labeling using commercially available agents, and site-specific RNA labeling

Chemical probes with biological activities

Cap analogs and chemical probes using in RNA labeling are great tools for studying the structures, locations, and possible functions of specific RNAs. In addition, a number of small molecules exhibit inhibitory effects on translation or control translation initiation and can be used as probes to study the translation process. Moreover, some of them have attracted much attention because of their good biological activity.

RNAs are highly sensitive to small changes in signal concentrations such as

Concluding remarks, future directions

RNA targets have been considered ‘unselectable’ or ‘undruggable’ for a long time. After years of effort, these limited examples highlight the challenges of specifically detecting RNA. Of course, there still has considerable progress made in the work of targeting RNA in recent years. There are several strategies for small molecules to regulate the translation process including directly targeting the RNAs involved in translation, modulating the activity of translation factors or sequence-specific

Acknowledgments

We thank the National Natural Science Foundation of China (Grants 81673286), the Guangdong Provincial Science and Technology Development Special Foundation (Public Interest Research and Capacity Building) (Grant 2016A020217006) for financial support of this study.

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