Multiple Novel Alternative Splicing Forms of FBXW7a Have a Translational Modulatory Function and Show Specific Alteration in Human Cancer Yueyong Liu 1. , Shancheng Ren 2. , Andres Castellanos-Martin 3 , Jesus Perez-Losada 3 , Yong-Won Kwon 1 , Yurong Huang 1 , Zeran Wang 1 , Mar Abad 4 , Juan J. Cruz-Hernandez 5 , Cesar A. Rodriguez 5 , Yinghao Sun 2 *, Jian-Hua Mao 1 * 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai, China, 3 Instituto de Biologı ´a Molecular y Celular del Ca ´ ncer (IBMCC), Instituto Mixto Universidad de Salamanca/CSIC, IBSAL, Campus Miguel de Unamuno s/n, Salamanca, Spain, 4 Deparment of Pathology, Hospital Universitario de Salamanca, IBSAL, Salamanca, Spain, 5 Department of Medical Oncology, Hospital Universitario de Salamanca, IBSAL, Salamanca, Spain Abstract FBXW7 acts as a tumor suppressor through ubiquitination and degradation of multiple oncoproteins. Loss of FBXW7 expression, which could be partially attributed by the genomic deletion or mutation of FBXW7 locus, is frequently observed in various human cancers. However, the mechanisms regulating FBXW7 expression still remain poorly understood. Here we examined the 59 region of FBXW7 gene to investigate the regulation of FBXW7 expression. We identified seven alternative splicing (AS) 59-UTR forms of FBXW7a that are composed of multiple novel non-coding exons. A significant difference in translational efficiency among these 59-UTRs variants was observed by in vivo Luciferase reporter assay and Western blot. Furthermore, we found that the mRNA level of the AS form with high translational efficiency was specifically reduced in more than 80% of breast cancer cell lines and in more than 50% of human primary cancers from various tissues. In addition, we also identified mutations of FBXW7 in prostate cancers (5.6%), kidney cancers (16.7%), and bladder cancers (18.8%). Our results suggest that in addition to mutation, differential expression of FBXW7a AS forms with different translational properties may serve as a novel mechanism for inactivation of FBXW7 in human cancer. Citation: Liu Y, Ren S, Castellanos-Martin A, Perez-Losada J, Kwon Y-W, et al. (2012) Multiple Novel Alternative Splicing Forms of FBXW7a Have a Translational Modulatory Function and Show Specific Alteration in Human Cancer. PLoS ONE 7(11): e49453. doi:10.1371/journal.pone.0049453 Editor: Jingwu Xie, Indiana University School of Medicine, United States of America Received August 19, 2012; Accepted October 9, 2012; Published November 14, 2012 Copyright: ß 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: JPL is partially supported by FEDER and MICINN (PLE2009-119), FIS (PI10/00328) ‘‘Fundacio ´ n Eugenio Rodrı ´guez Pascual’’, and Fundacio ´ n Inbiomed (Instituto Oncolo ´ gico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa). YS is supported by the National Basic Research Program of China (2012CB518300) and the Ministry of Science & Technology of Shanghai (08410701500). JHM is supported by the National Institutes of Health, National Cancer Institute grant R01 CA116481, the Low Dose Scientific Focus Area, Office of Biological & Environmental Research, US Department of Energy (DE-AC02-05CH11231), Laboratory Directed Research & Development Program (LDRD), and NASA Specialized Center for Research in Radiation Health Effects (NNX09AM52G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sunyh@medmail.com.cn (YS); JHMao@lbl.gov (J-HM) . These authors contributed equally to this work. Introduction The FBXW7 gene is a transcriptional target of p53, whose expression is upregulated in a p53-dependent-manner after radiation treatment [1]. The FBXW7 gene encodes an F-box protein, which is essential for the ubiquitination of different oncoproteins, including c-Myc [2,3], c-Jun [4], cyclin E [5,6], different members of the Notch family [7,8], Aurora-A [1,9], mTor [10,11], and KLF5 [12,13]. Overexpression of several of these targets, such as cyclin E [14], c-Myc [15] and Aurora-A [16] has been implicated to induce genomic instability. These observations demonstrated that FBXW7 is a human tumor suppressor gene, a conclusion that is also supported by the discovery of FBXW7 gene mutations in cancers from a wide spectrum of human tissues, such as bile duct, blood, bone, brain, breast, colon, endometrium, stomach, lung, ovary, pancreas, and prostate, with overall 6% point mutation frequency [17]. Deletion of the Fbxw7 gene in mice leads to embryonic lethality, but heterozygous mice develop normally [18,19]. Although they do not develop spontaneous tumors, radiation exposure gives rise to different types of tumors, including a range of epithelial cancers [1]. Mice that carry inactivated alleles of both Fbxw7 and p53 show acceleration of tumor development. Haploinsufficient loss of Fbxw7 is observed in most lymphomas in this mouse model, even those arising from Fbxw7/p53 double heterozygous mice, i.e., loss of only one copy of the gene can generate a substantial biological impact [1]. Similar observations of heterozygous mutations were subsequently found in human tumors [20]. It is therefore likely that the overall impact of this tumor suppressor gene in human cancer is greater than the 6% point mutation frequency mentioned above, since loss of only one gene copy can have a substantial effect on tumor development. Deletions of chromosome 4q31, on which FBXW7 is located, are common in many types of human PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49453