Evidence that the Upf1-related molecular motor scans the 3′-UTR to ensure mRNA integrity

Published online 2 May 2012 Nucleic Acids Research, 2012, Vol. 40, No. 14 6887–6897 doi:10.1093/nar/gks344 Evidence that the Upf1-related molecular motor scans the 30-UTR to ensure mRNA integrity Toshiaki Shigeoka, Sayaka Kato, Masashi Kawaichi and Yasumasa Ishida* Division of Gene Function in Animals, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan Received September 21, 2011; Revised April 6, 2012; Accepted April 11, 2012 ABSTRACT Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation–terminating ribosome and then with a downstream exon– junction complex (EJC), which is deposited at exon–exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon–exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 30 -untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 50 -side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation–termination codon and a downstream exon– exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 30 -UTR in the 50 -to-30 direction for the mRNA-binding factors including EJCs to ensure mRNA integrity. INTRODUCTION RNA helicases comprise a large family of enzymes that are thought to unwind double-stranded RNA molecules through the hydrolysis of NTP and participate in a variety of essential physiological processes. Several members of the family have been demonstrated in vitro to unwind RNA duplexes and translocate unidirectionally as processive molecular motors along the RNA track (1). Despite tremendous biochemical studies that revealed much about these enzymatic properties, the mechanisms through which RNA helicases exert influences on a variety of physiological processes are poorly understood. Upf1 is a highly conserved RNA helicase that is essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins (2,3). NMD has evolved to recognize and specifically degrade aberrant mRNAs whose open reading frames (ORFs) are truncated by the presence of premature termination codons (PTCs) (3,4). NMD is important not only for the suppression of mutated gene expression, but also for the regulation of physiological gene function during development and homeostasis [reviewed in (5)]. The NMD pathway is activated through a translation-dependent mechanism, in which trans-acting factors including Upf1 are recruited to a translation–terminating ribosome (6,7). Previous studies demonstrated that the purified Upf1 protein exhibits the RNA-dependent ATPase activity that leads to the unwinding of RNA duplexes in vitro (8–10), and mutations affecting its ATPase/helicase activity impair NMD (8,11). Although a recent report indicates that the helicase activity of Upf1 promotes disassembly of mRNP undergoing the final mRNA-degradation steps of NMD (12), the roles and substrates of the Upf1 helicase activity during the induction phase of NMD still remain unkonwn. In vertebrates, a translation–termination codon located >50–55 nt upstream of an exon–exon junction is generally recognized as premature (13,14), based on the crucial function of the exon–junction complex (EJC) (15,16). The EJC is deposited during splicing at 20–24 nt upstream of each exon–exon junction as the signature of intron removal (15,16) and displaced from an mRNA by a translating ribosome in the cytoplasm (17,18). While all *To whom correspondence should be addressed. Tel: +81 743 72 5531; Fax: +81 743 72 5539; Email: Present addresses: Toshiaki Shigeoka, Department of Stem Cell and Developmental Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu-shi, Mie 514-8507, Japan. Sayaka Kato, ASPION Corporation, 7-1-17 Minatojima Minami-machi, Chuo-ku, Kobe 650-0047, Japan. ß The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 6888 Nucleic Acids Research, 2012, Vol. 40, No. 14 EJCs are removed from normal mRNAs during the first round of translation, the PTC-containing mRNAs, in which the ribosome does not pass all of the exon–exon junctions, remain associated with EJCs during the translation–termination process (17,18). According to the current model of the EJC-dependent NMD, Upf1 is first recruited to a translation–terminating ribosome as a component of the SMG1-Upf1-eukaryotic release factor 3 (eRF3)–eRF1 (SURF) complex, and the subsequent interaction between the Upf1-containing SURF complex and the downstream EJCs, with which two of the other essential NMD factors Upf2 and Upf3 are associated, activates the mRNA-degradation pathway (7,10,19). Several lines of evidence indicate that the interaction between the SURF complex and the EJC is a highly efficient and accurate process. Even if the distance between a PTC and a downstream exon–exon junction is extremely long (e.g. 1–5 kb), the mRNA undergoes NMD, indicating the presence of a mechanism to bridge the spatial gap between a translation–termination site and a downstream exon–exon junction on an mRNA molecule (20,21). In this article, we show that stable RNA secondary structures inserted between a PTC and a downstream exon–exon junction operate as cis-acting elements to increase the levels of potential NMD targets. We also demonstrate that the general translation–termination processes induce the binding of Upf1 to mRNAs containing a stable secondary structure within the 30 -untranslated regions (UTRs) in a splicing-independent manner and reveal the accumulation of the Upf1-related molecule(s) at the 50 -side of the secondary structure. We further show that the helicase activity of Upf1 is required for the activation of NMD only when a PTC is located significantly apart from a downstream exon–exon junction. Based on these findings, we propose a model, in which Upf1 acts as a helicase-driven molecular motor that translocates along the 30 -UTR of an mRNA molecule in the 50 -to-30 direction, scanning for the mRNA-binding (...truncated)