Destabilisation of Argonaute 2 generates a truncated protein: halfAgo2

Abstract

The Argonaute 2 (Ago2) protein is an essential effector protein in miRNA-mediated mechanisms that regulate gene expression. Ago2 directly binds to the miRNA, forming the RISC. RISC function is critical to controlling key biological processes and when dysregulated can result in disease pathogenesis. Understanding Ago2 protein stability and turnover will further our understanding in how RISC function is regulated. In human cells, we discovered a previously unidentified ~55 kDa protein that is a truncated form of Ago2, that is formed from proteolytic cleavage of the full length Ago2 protein. Further experiments are needed to determine (i) the detailed mechanism that forms halfAgo2 (ii) the cellular or environmental triggers or stresses that initiate halfAgo2 production and (iii) if halfAgo2 has a potentially new role in gene regulation. Introduction miRNAs are endogenous small molecules that are essential regulators of human development. miRNAs function by targeting the post-transcriptional stages of gene expression, via several distinct mechanisms [1]. miRNA regulatory function depends on the miRNA directly binding to an Argonaute (Ago) protein, forming the RNA Induced Silencing Complex (RISC). In this complex the miRNA acts a guide, by binding to a complementary site within mRNA and bringing the RISC, and its associated regulatory proteins, to the target [2] [3]. RISC regulates key biological processes, therefore any disruption to RISC function can have severe consequences, and misregulation of RISC is implicated in the development of disease [4]. It is therefore important that the stability of RISC and accordingly its components, miRNA andAgo, are controlled. There are several knownmechanisms that mediatemiRNA stability and turnover, including homeostatic and feedbackmechanisms that coordinate miRNA levels with Ago levels [5] [6] [7] [8]. However, we have only limited understanding into mechanisms that regulate the turnover of Ago proteins and the RISC complex. There is emerging evidence that Ago function and stability is mediated by a variety of post-translational modifications of the protein, which occurs as a consequence of complex signalling pathways [9] [10] [11] [12] [13]. These modifications can alter protein function, stability, and localisation. Depending on the modification, these changes can be permanent or reversible. Therefore Ago levels are potentially highly dynamic and are responsive to internal and external stimuli. While the 4 human Ago proteins (1–4) display some functional redundancy, Ago2 is the most abundant in commonly used human cell lines [14] and the most studied in miRNA regulation. Objective While analysing the protein expression of full-length (FL), endogenous, Ago2 (~85 kDa) in HeLa cell lysate, we observed that an Ago2-specific monoclonal antibody targeted to the N-terminus of the protein [15], also bound to a previously unidentified protein of approximately 55 kDa. The objective of this study was to investigate this 55 kDa protein, and to test our hypothesis that this protein is a truncated form of FL human Ago2. Destabilisation of Argonaute 2 generates a truncated protein: halfAgo2 DOI: N/A Matters (ISSN: 2297-8240) | 2 a