Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) Leigh-Ann MacFarlane, Paul Murphy Dalhousie University, Department of Physiology and Biophysics, Faculty of Medicine, 5850 College Street Sir Charles Tupper Medical Building, Halifax, Nova Scotia B3H 1X5, Canada (LAM, PM)

Published in Atlas Database: April 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/NUDT6ID41593ch4q28.html DOI: 10.4267/2042/44935 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity DNA/RNA Other names: ASFGF2, bFGF, FGF-2, FGF-AS, Note FGF2AS, gfg, gfg-1 Human NUDT6 is located on 4 in the HGNC (Hugo): NUDT6 region of q28 on the reverse strand, opposite to FGF-2 Location: 4q28.1 locus. FGF-2 and NUDT6 overlap at 3' Note: NUDT6 is a novel nudix with unknown ends, and the mRNAs form a sense-antisense pair. The function. NUDT6 mRNA (referred to as FGF-AS) has been implicated in the regulation of FGF2 mRNA stability.

Figure A. The schematic representation of the overlap between human NUDT6 (FGF-AS) and FGF2 gene transcripts (colored boxes, coding region; connecting vertical lines, complementary regions between transcripts). Adapted from: MacFarlane LA, et al., 2010. Molecular Endocrinology 24.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 29 NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) MacFarlane LA, Murphy P

Figure B. The schematic representation of the human NUDT6 gene transcripts, variants a-g (red boxes, coding region; yellow boxes, untranslated region).

Description Transcription The human NUDT6 gene is 34271 bp in length, The primary transcript can be alternatively spliced to composed of a 5'UTR, 16 exons, 6 introns and a produce at least 7 splice variants, a-d. Full length 3'UTR. The 5' and 3'UTR contain a variety of variants a and b only differ in the use of an alternative regulatory elements that regulate NUDT6 expression. first exon, designated 1A or 1B. The 3' ends of the NUDT6 gene transcription is regulated by a core variants share sequence similarity. Two transcriptional promoter mapping from -1871 to +181 (relative to the start sites have been identified, one 15 bp upstream of transcription start site +1, up-stream -), which is 44 kb the 1A exon (designated +1) and the other 84 bp downstream from the FGF-2 promoter, however the upstream of the 1B exon (+312). It is unclear whether proximal -151/+181 region confers almost full another transcription start site specific for other transcription activity. variants are located further downstream. The promoter lacks a consensus TATA box or CCAAT NUDT6 transcripts are often designated FGF-AS (FGF element. The region between the first two exons antisense). The two longest transcripts, a and b, are contains two Sp1 transcription factor binding sites (- classified as cis-antisense because they are transcribed 372/-58 relative to the first exon start site). The from the same gene locus, on the opposite DNA strand common upstream region (all subsequent positions and their 3'UTR is fully complementary to the 3'UTR relative to first exon start site) from these start sites of FGF-2 over two regions 583 bp and 56 bp in length. contains a multitude of tissue specific transcription Pseudogene factor binding sites, which include lymphocyte specific factors Ets at -229 and -83, GATA at -662 and +56, NA. Lyf-1 at -981; skeletal muscle consensus E-boxes at - 901 and +30; cardiac factor Nkx-2.5 at -1501 and -582; Protein liver and adipose C/EBP factor at -624; and testis Note specific factors SRY at -1740, -671, +163, +171 and Human NUDT6 encodes 3 novel nudix with Sox-5 at -1472, -632. unknown function. Two negative regulatory elements also reside in this shared upstream region, at -1871 and -1315. The Description NUDT6 3'UTR contains a singe AU-rich element NUDT6 splice variants a-c contain open reading frames (ARE) and seven AU-rich-like sequences which (ORF) that predict isoforms of a novel nudix motif negatively regulate mRNA stability. A portion of the protein, originally designated GFG. The nudix box NUDT6 coding region and 3'UTR (+531/+1167) is motif is defined by the consensus signature amino acid fully complementary to the 3'UTR of FGF2 and sequence interaction through this region leads to the formation of GX5EX7REUXEEXGU, a sense-antisense pair.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 30 NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) MacFarlane LA, Murphy P

Schematic representing predicted NUDT6 isoforms encoded by alternate splice RNA transcripts (deep blue boxes, nudix motif; light blue box, MTSP-mitochondrial targeting signal peptide). where X is any amino acid and U is a bulky pituitary expresses moderate levels of NUDT6 and no hydrophobic amino acid, usually isoleucine, leucine or FGF-2 while pituitary tumors have reduced NUDT6 valine. To date, three different molecular weight expression and high levels of FGF-2. The isoforms have been identified in human, of 35, 28 and NUDT6/FGF-2 expression ratio decreases dramatically 17 kDa, which are presumably generated by alternative in tumors compared to normal tissue. Varying translation initiation. Isoforms are designated as a, b or NUDT6/FGF-2 ratios have also been observed in c however, this does not necessarily indicate that the esophageal adenocarcinomas. Additionally, transient isoform was synthesized from the corresponding increase in NUDT6 expression occurs in response to transcript variant. The 35 kDa isoform is synthesized treatment with interleukin-2 and prolactin. from the full length FGF-ASa, by translation initiation Localisation from the in-frame AUG codon located in exon 1A. The origin of the 28 kDa isoform is unclear. It is suspected NUDT6 can reside in the mitochondria, cytoplasm and that it is synthesized from an in-frame CUG codon in nucleus, however its subcellular localization varies exon 2 of either FGF-AS a or b. with isoform, cell type, disease state and extracellular However, it is possible that the 28 kDa product is a stimulus. NUDT6a predominantly localizes to proteolytic fragment of the 35 kDa isoform. The 17 mitochondria whereas NUDT6b and NUDT6c kDa isoform may arise from translation initiation at an primarily reside in the cytoplasm and nucleus. NUDT6 in-frame CUG codon in exon 3 of FGF-ASb or AUG is only found in the cytoplasm of normal esophageal codon in the first exon of FGF-ASc. The NUDT6 squamous epithelial cells whereas in normal isoforms are detected as stable homo- and hetero- lymphocytes it is exclusively nuclear. However, dimers by western blotting, which can be disrupted by transformation of these cells results in the redistribution dithiothreitol (DTT) and boiling. Potential dimerization of NUDT6. Cells from esophageal adenocarcinoma domains have been mapped to both the N-terminus and tumors and lymph nodes of patient with immunoblastic COOH-terminus of NUDT6. lymphoma localize NUDT6 to the nucleus and cytoplasm. Expression Function NUDT6 is expressed in a tissue and developmental stage specific manner. RNA transcripts are detected in The RNA and protein products appear to have distinct most human tissues including liver, thymus, spleen, biological functions. NUDT6 mRNA (FGF-AS) plays a peripheral blood leukocytes, heart skeletal muscle, role in FGF-2 regulation, proliferation, and cell testis, colon and kidney. However, which transcript survival. Additionally, the NUDT6 protein has been variants are expressed appears to be tissue specific. The implicated in the control of hormone production in the full length FGF-ASb is thought to be the predominant pituitary, and possibly in the removal of potentially variant in most tissues however variant FGF-ASa is the hazardous compounds and metabolites by virtue of its major variant in normal hematopoietic tissues. conserved nudix domain. However, it is not always Furthermore, some tissues co-express FGF-2 and the clear whether a specific action is a result of the RNA or ratio between FGF-2 and FGF-AS transcripts varies protein function and this is further complicated by with tissue and development stage. FGF-AS levels are multiple antisense splice variants and protein isoforms. relatively low in many embryonic tissues, with FGF-AS regulates FGF-2 transcript stability. Although expression increasing dramatically in a tissue specific the details of the mechanism involved are unclear, manner postnatally. FGF-2 and FGF-AS exhibit an evidence suggest involvement of RNA interference inverse relationship in normal tissues, tumor cell lines, and/or a dsRNA duplex formed between the 3'UTRs of embryonic development and throughout cell cycle FGF-AS and FGF-2. In addition to regulating FGF-2 progression. abundance it has been suggested that it also controls The level of NUDT6 expression and its ratio with FGF- FGF-2 isoform translation and localization. The 2 expression is frequently altered in tumors. Normal regulatory role of FGF-AS over FGF-2 is thought to

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 31 NUDT6 (nudix (nucleoside diphosphate linked moiety X)-type motif 6) MacFarlane LA, Murphy P

account for observed effects on cell proliferation and Melanoma survival. Note NUDT6 protein is a nudix hydrolase which is a class of Melanoma is a malignant tumor of melanocytes, which "house cleaning" enzymes capable of hydrolyzing a are found primarily in the skin, however they can broad range of substrates, all defined as nucleoside develop in melanocytes found in the eye and bowel. A diphosphates linked to some other moiety, that include characteristic of aggressive melanomas is their ability nucleoside di- and triphosphates, dinucleoside and to form fluid-conducting vasculogenic-like networks. diphosphoinositol polyphosphates, nucleotide sugars and RNA caps. The substrate of human NUDT6 has yet Oncogenesis to be elucidated and therefore its physiological function A preliminary study investigating these 3D tubular remains unknown. NUDT6 has observed effects on cell networks within tumors identified NUDT6 as one of proliferation independent of those associated with FGF- the many genes overexpressed in aggressive AS. NUDT6 overexpression in human colorectal melanomas and speculated it is involved in promoting cancer cells increases proliferation. Perhaps NUDT6's self-renewal and tumor cell plasticity in melanoma effects on proliferation are dependent on expression cancer networks. Additionally, they suggest FGF-AS level, isoform and/or cell type, as is the case with FGF- could play a role in the development of the endothelia- 2. Furthermore, NUDT6 is involved in hormone lined vasculature networks in melanomas indirectly production. GFG expression can increase levels of through its regulatory control over FGF-2 expression, prolactin. However it is unclear if these effects are which is associated with angiogenesis, proliferation and mediated through the same MAPK pathway utilized by survival. FGF-2 to increase prolactin expression. Colorectal cancer Additionally, NUDT6 alters the isoform ratio of growth Note hormone, by increasing synthesis of the 22 kDa Colorectal cancer refers to uncontrolled growth of cells isoform and not the 20 kDa. that line the colon, rectum and appendix, collectively Homology the large intestine. NUDT6 contains a conserved nudix motif common to Oncogenesis other members of the Nudix family of Induced overexpression of NUDT6 in a variety of phosphohydrolases. The Nudix motif is human colorectal cells significantly increases cancer GXXXXXEXXXXXXXREUXEEXGU where U is cell proliferation and their clonogenic capacity. Isoleucine, Leucine, or Valine and X is any amino acid. NUDT6 is described as having tumor promoting NUDT6 is highly conserved among man, cow, mouse, functions in this cellular environment and it is worm, and fruit fly, and GFG homologs across species suggested that it plays a role in colorectal cancer are more evolutionarily related to each other than to development and progression. other nudix proteins from the same species. Endometriosis Implicated in Note Endometriosis is a medical condition affecting the Esophageal adenocarcinoma endometrium lining of the uterus. The endometrium is comprised of hormonally responsive cells that Note proliferate and secrete under the influence of estrogen Esophageal adenocarcinoma refers to uncontrolled and progesterone. Upon menstruation the endometrium growth of glandular cells in the esophagus and the lining is shed as a part of the menstrual flow. junction between the esophagus and the stomach. Endometriosis describes the presence of endometrial Prognosis cells outside of the uterus, such as the ovaries, fallopian Elevated expression of FGF-AS in FGF-2 expressing tubes, bladder and interstitial space in the abdominal esophageal adenocarcinomas is associated with reduced cavity. tumor reoccurrence following surgical resection of Patients with endometriosis lesions have reduced FGF- tumors and increased survival rates, suggesting that it AS-b mRNA levels and elevated FGF-2 mRNA levels may be used as a prognostic indicator. during the late proliferative phase of the menstrual Oncogenesis cycle, compared to control patients. This increased Esophageal adendocarcinoma tumors overexpressed FGF/FGF-AS ratio is thought to contribute to the FGF-AS and cytoplasmic GFG in comparison to development of endometriosis. normal match esophageal tissue. However, the reduced tumor reoccurrence and improved survival rates References specifically correlated to FGF-AS levels, not GFG levels. Evidence suggests FGF-AS tumor suppressive Knee RS, Pitcher SE, Murphy PR. Basic fibroblast growth factor sense (FGF) and antisense (gfg) RNA transcripts are role is a result of its post-transcription control over expressed in unfertilized human oocytes and in differentiated FGF-2. adult tissues. Biochem Biophys Res Commun. 1994 Nov 30;205(1):577-83

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Murphy PR, Knee RS. Identification and characterization of an Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, antisense RNA transcript (gfg) from the human basic fibroblast Porter GA, Veugelers PJ, Murphy PR, Casson AG. Basic growth factor gene. Mol Endocrinol. 1994 Jul;8(7):852-9 fibroblast growth factor (FGF-2) overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which Bessman MJ, Frick DN, O'Handley SF. The MutT proteins or is ameliorated by coexpression of the FGF-2 antisense gene. "Nudix" hydrolases, a family of versatile, widely distributed, Clin Cancer Res. 2005 Nov 1;11(21):7683-91 "housecleaning" enzymes. J Biol Chem. 1996 Oct 11;271(41):25059-62 Zhang SC, Barclay C, Alexander LA, Geldenhuys L, Porter GA, Casson AG, Murphy PR. Alternative splicing of the FGF Knee R, Murphy PR. Regulation of gene expression by natural antisense gene: differential subcellular localization in human antisense RNA transcripts. Neurochem Int. 1997 tissues and esophageal adenocarcinoma. J Mol Med. 2007 Sep;31(3):379-92 Nov;85(11):1215-28 Gagnon ML, Moy GK, Klagsbrun M. Characterization of the Demou ZN, Hendrix MJ. Microgenomics profile the promoter for the human antisense fibroblast growth factor-2 endogenous angiogenic phenotype in subpopulations of gene; regulation by Ets in Jurkat T cells. J Cell Biochem. 1999 aggressive melanoma. J Cell Biochem. 2008 Oct 1;105(2):562- Mar 15;72(4):492-506 73 Asa SL, Ramyar L, Murphy PR, Li AW, Ezzat S. The MacFarlane LA, Gu Y, Casson AG, Murphy PR. Regulation of endogenous fibroblast growth factor-2 antisense gene product fibroblast growth factor-2 by an endogenous antisense RNA regulates pituitary cell growth and hormone production. Mol and by argonaute-2. Mol Endocrinol. 2010 Apr;24(4):800-12 Endocrinol. 2001 Apr;15(4):589-99 Sukhthankar M, Choi CK, English A, Kim JS, Baek SJ. A Mihalich A, Reina M, Mangioni S, Ponti E, Alberti L, Viganò P, potential proliferative gene, NUDT6, is down-regulated by Vignali M, Di Blasio AM. Different basic fibroblast growth factor green tea catechins at the posttranscriptional level. J Nutr and fibroblast growth factor-antisense expression in eutopic Biochem. 2010 Feb;21(2):98-106 endometrial stromal cells derived from women with and without endometriosis. J Clin Endocrinol Metab. 2003 Jun;88(6):2853- This article should be referenced as such: 9 MacFarlane LA, Murphy P. NUDT6 (nudix (nucleoside Baguma-Nibasheka M, Li AW, Osman MS, Geldenhuys L, diphosphate linked moiety X)-type motif 6). Atlas Genet Casson AG, Too CK, Murphy PR. Coexpression and regulation Cytogenet Oncol Haematol. 2011; 15(1):29-33. of the FGF-2 and FGF antisense genes in leukemic cells. Leuk Res. 2005 Apr;29(4):423-33

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