Aspergillus nidulans Upf1: putative role of conserved active sites in ribosome recycling and 3′ end mRNA tagging

BioscienceHorizons Volume 8 2015  10.1093/biohorizons/hzv004 Research article Aspergillus nidulans Upf1: putative role of conserved active sites in ribosome recycling and 3′ end mRNA tagging Ryan Langley* *Corresponding author: 45 The Spinney, Finchfield, Wolverhampton WV3 9EU, England. Email: Supervisor: Dr Igor Morozov, Faculty of Health and Life Sciences, Coventry University, Coventry, England. Up-frameshift protein 1 (Upf1) is a multidomain RNA helicase that is conserved from yeast to humans. Upf1 is critical for nonsense-mediated decay (NMD), a quality control mechanism that detects and eliminates aberrant transcripts harbouring a premature termination codon (PTC) and thus plays an important role in maintaining the fidelity of gene expression. Additionally, Upf1 is implicated in a broad range of cellular responses from chromosome maintenance to mRNA degradation and translational repression. Recent findings show that Upf1 also triggers 3′ end mRNA tagging, the addition of non-templated pyrimidines (C/U) to the 3′ end of adenylated and non-adenylated (histone) mRNAs. 3′ end tagging is seen as a general precursor of mRNA degradation and has been found to occur in fungi, plants and mammals. In Aspergillus nidulans, 3′ end tagging of normal and aberrant transcripts containing PTCs occurs in an Upf1-dependent manner. Intriguingly, tagging of transcripts harbouring PTCs is not essential for transcript degradation as the disruption of either of the two enzymes that mediate 3′ end RNA tagging, CutA and CutB results in decreased efficiency of ribosome dissociation from the PTC. However, the exact role of Upf1 and its functional domains in inducing tagging and ribosome dissociation remains unknown. Therefore, the aim of this work is to propose a model for the detailed mutational analysis of A. nidulans Upf1 in relation to its role in triggering 3′ end tagging and translation termination. From published data of mutation analysis, active site residues of the yeast and human Upf1 proteins have been identified and aligned to their A. nidulans homologue. Analysis of the structural organization of A. nidulans Upf1 reveals the presence of two major conserved domains and a number of putative actives site residues which may be crucial for 3′ end mRNA tagging, translational repression and ribosome termination. The importance of a greater understanding of the role of Upf1 in regulation of gene expression in A. nidulans, a model organism for Aspergillus species of medical and industrial importance, is discussed. Key words: upf1, 3′ end tagging, Aspergillus, nonsense-mediated decay Submitted on 18 June 2014; accepted on 30 April 2015 Introduction Gene expression comprises multiple stages and revolves around RNA, from transcription to RNA processing and translation. The cellular machinery that constitutes to each stage is often complex, and therefore, errors may be introduced into RNA molecules at any step of its biogenesis (Morozov and Caddick, 2012). To minimize the impact of such inaccuracies and ensure the fidelity of gene expression, cells have evolved a number of quality control mechanisms that detect and eliminate transcripts that are no longer functional (e.g. aberrant mRNAs) or potentially harmful (e.g. malfunctional non-coding RNAs). These quality control mechanisms come in a form of multiple mRNA decay pathways and can require © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons 1 Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Faculty of Health and Life Science, Department of Applied Sciences and Health, Coventry University, Priory Street, Coventry CV1 5FB, England Research article active translation (Doma and Parker, 2007; Shoemaker and Green, 2012). One such pathway is nonsense-mediated decay (NMD) in which a premature termination codon (PTC), caused by nonsense mutations or frame-shifts, is recognized by translating ribosomes, triggering a cascade of events leading to efficient degradation of the PTC+ mRNA and thus preventing the accumulation of potentially harmful truncated proteins (Imamachi, Tani and Akimitsu, 2012; Popp and Maquat, 2013). Upf1 is a highly conserved RNA helicase within eukaryotes, from yeast to humans (Applequist et al., 1997). It is a principal NMD factor involved in the recognition and degradation of defective mRNA harbouring a PTC. Upf1 is an intriguing protein which in addition to NMD has been implicated in a broad range of processes, including development, chromosome maintenance, cell cycle progression and DNA replication (Imamachi, Tani and Akimitsu, 2012). Recent work has discovered that Upf1 plays a crucial role in 3′ end tagging of physiological and PTC+ transcripts in mammals and Aspergillus nidulans, namely the non-canonical addition of U/C and possibly G nucleotides to the 3′ end of mRNA (Mullen and Marzluff, 2008; Rissland and Norbury, 2009; Morozov et al., 2010a, 2012; Chang et al., 2014). 3′ end tagging appears to be a conserved eukaryotic surveillance mechanism in mammalian, plant and fungi cells with Saccharomyces cerevisiae being the prominent exception. Upf1-mediated C/U tagging appears to be the point at which mRNA is earmarked for subsequent translational suppression and degradation, while G tagging is mainly associated with a long poly(A) tail of mRNA in mammals (Chang et al., 2014) and A. nidulans (Morozov and Caddick, personal communication) and potentially acts as a defence pathway against mRNA degradation. However, the exact mechanism of 3′ end tagging and its role in the regulation of gene expression is not yet understood. These data underline the importance of Upf1 beyond NMD. Thus, it would be of great importance to identify active sites and conduct mutational analysis of Upf1 in A. nidulans to understand the mechanism by which Upf1 regulates mRNA 3′ end tagging and subsequent ribosome recycling of both physiological and aberrant transcripts. 2 In this study, two conserved domains in A. nidulans Upf1 (AN0646) have been identified. By reviewing published data of mutation analysis of Upf1 in yeast and mammalian systems, numerous putative actives sites that may be implicated in 3′ end mRNA tagging, translational repression and ribosome termination were also found. Detailed mutation analysis of A. nidulans Upf1 with respect to its role in triggering 3′ end mRNA tagging and translation termination has been proposed. These results and their potential application in the mutational analysis of Upf1 Aspergillus species of industrial and medical importance are discussed. The novel role of Upf1 in 3′ tagging in A. nidulans In eukaryotes, cytoplasmic 3′ tagging of RNA, primarily by the addition of non-templated pyrimidines (C/U), is a general precursor to RNA degradation (Morozov and Caddi (...truncated)