Zebrafish Adar2 Edits the Q/R Site of AMPA Receptor Subunit gria2α Transcript to Ensure Normal Development of Nervous System and Cranial Neural Crest Cells
Background Adar2 deaminates selective adenosines to inosines (A-to-I RNA editing) in the double-stranded region of nuclear transcripts. Although the functions of mouse Adar2 and its biologically most important substrate gria2, encoding the GluA2 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor, have been extensively studied, the substrates and functions of zebrafish Adar2 remain elusive. Methods/Principal Findings Expression of Adar2 was perturbed in the adar2 morphant (adar2MO), generated by antisense morpholio oligonucleotides. The Q/R editing of gria2α was reduced in the adar2MO and was enhanced by overexpression of Adar2, demonstrating an evolutionarily conserved activity between zebrafish and mammalian Adar2 in editing the Q/R site of gria2. To delineate the role of Q/R editing of gria2α in the developmental defects observed in the adar2MO, the Q/R editing of gria2α was specifically perturbed in the gria2αQRMO, generated by a morpholio oligonucleotide complementary to the exon complementary sequence (ECS) required for the Q/R editing. Analogous to the adar2-deficient and Q/R-editing deficient mice displaying identical neurological defects, the gria2αQRMO and adar2MO displayed identical developmental defects in the nervous system and cranial cartilages. Knockdown p53 abolished apoptosis and partially suppressed the loss of spinal cord motor neurons in these morphants. However, reducing p53 activity neither replenished the brain neuronal populations nor rescued the developmental defects. The expressions of crestin and sox9b in the neural crest cells were reduced in the adar2MO and gria2αQRMO. Overexpressing the edited GluA2αR in the adar2MO restored normal expressions of cresting and sox9b. Moreover, overexpressing the unedited GluA2αQ in the wild type embryos resulted in reduction of crestin and sox9b expressions. These results argue that an elevated GluA2αQ level is sufficient for generating the cranial neural crest defects observed in the adar2MO. Our results present a link between dysfunction of AMPA receptors and defective development of the nervous system and cranial neural crest in the zebrafish.
References
[1]
Bass BL (2002) RNA editing by adenosine deaminases that act on RNA. Annu Rev Biochem 71: 817–846. doi: 10.1146/annurev.biochem.71.110601.135501
[2]
Cenci C, Barzotti R, Galeano F, Corbelli S, Rota R, et al. (2008) Down-regulation of RNA editing in pediatric astrocytomas: ADAR2 editing activity inhibits cell migration and proliferation. J Biol Chem 283: 7251–7260. doi: 10.1074/jbc.m708316200
[3]
Nishikura K (2010) Functions and regulation of RNA editing by Adar deaminases. Annu Rev Biochem 79: 321–349. doi: 10.1146/annurev-biochem-060208-105251
[4]
Dominissini D, Moshitch-Moshkovitz S, Amariglio N, Rechavi G (2011) Adenosine-to-inosine RNA editing meets cancer. Carcinogenesis 32: 1569–1577. doi: 10.1093/carcin/bgr124
[5]
Keegan LP, Gallo A, O'Connell MA (2001) The many roles of an RNA editor. Nat Rev Genet 2: 869–878. doi: 10.1038/35098584
[6]
Heale BS, Keegan LP, McGurk L, Michlewski G, Brindle J, et al. (2009) Editing independent effects of ADARs on the miRNA/siRNA pathways. EMBO J 28: 3145–3156. doi: 10.1038/emboj.2009.244
[7]
Hartner JC, Schmittwolf C, Kispert A, Muller AM, Higuchi M, et al. (2004) Liver disintegration in the mouse embryo caused by deficiency in the RNA-editing enzyme ADAR1. J Biol Chem 279: 4894–4902. doi: 10.1074/jbc.m311347200
[8]
Higuchi M, Maas S, Single FN, Hartner J, Rozov A, et al. (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406: 78–81.
[9]
Sommer B, K？hler M, Sprengel R, Seeburg PH (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67: 11–19. doi: 10.1016/0092-8674(91)90568-j
[10]
Brusa R, Zimmermann F, Koh DS, Feldmeyer D, Gass P, et al. (1995) Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 270: 1677–1680. doi: 10.1126/science.270.5242.1677
[11]
Palladino MJ, Keegan LP, O'Connell MA, Reenan RA (2000) A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell 102: 437–449. doi: 10.1016/s0092-8674(00)00049-0
[12]
Wang Q, Miyakoda M, Yang W, Khillan J, Stachura DL, et al. (2004) Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. J Biol Chem 279: 4952–4961. doi: 10.1074/jbc.m310162200
[13]
Slavov D, Crnogorac-Jurcevic T, Clark M, Gardiner K (2000) Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish. Gene 250: 53–60. doi: 10.1016/s0378-1119(00)00175-x
[14]
Slavov D, Gardiner K (2002) Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns. Gene 299: 83–94. doi: 10.1016/s0378-1119(02)01016-8
[15]
Kung SS, Chen YC, Lin WH, Chen CC, Chow WY (2001) Q/R RNA editing of the AMPA receptor subunit 2 (GRIA2) transcript evolves no later than the appearance of cartilaginous fishes. FEBS Lett 509: 277–281. doi: 10.1016/s0014-5793(01)03183-0
[16]
Lin W-H, Wu C-H, Chen Y-C, Chow W-Y (2006) Embryonic expression of zebrafish AMPA receptor genes: zygotic gria2α expression initiates at the midblastula transition. Brain Res 1110: 46–54. doi: 10.1016/j.brainres.2006.06.054
[17]
Chen YC, Kao SC, Chou HC, Lin WH, Wong FH, et al. (2008) A real-time PCR method for the quantitative analysis of RNA editing at specific sites. Anal Biochem 375: 46–52. doi: 10.1016/j.ab.2007.12.037
[18]
Hoppmann V, Wu JJ, Soviknes AM, Helvik JV, Becker TS (2008) Expression of the eight AMPA receptor subunit genes in the developing central nervous system and sensory organs of zebrafish. Dev Dyn 237: 788–799. doi: 10.1002/dvdy.21447
[19]
Wahlstedt H, Daniel C, Enstero M, Ohman M (2009) Large-scale mRNA sequencing determines global regulation of RNA editing during brain development. Genome Res 19: 978–986. doi: 10.1101/gr.089409.108
[20]
Hideyama T, Yamashita T, Suzuki T, Tsuji S, Higuchi M, et al. (2010) Induced loss of ADAR2 engenders slow death of motor neurons from Q/R site-unedited GluR2. J Neurosci 30: 11917–11925. doi: 10.1523/jneurosci.2021-10.2010
[21]
Chen J, Ruan H, Ng SM, Gao C, Soo HM, et al. (2005) Loss of function of def selectively up-regulates Delta113p53 expression to arrest expansion growth of digestive organs in zebrafish. Genes Dev 19: 2900–2911. doi: 10.1101/gad.1366405
[22]
Langheinrich U, Hennen E, Stott G, Vacun G (2002) Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. Curr Biol 12: 2023–2028. doi: 10.1016/s0960-9822(02)01319-2
[23]
Danilova N, Kumagai A, Lin J (2010) p53 upregulation is a frequent response to deficiency of cell-essential genes. PloS one 5: e15938. doi: 10.1371/journal.pone.0015938
[24]
Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, et al. (2007) p53 activation by knockdown technologies. PLoS Genet 3: e78. doi: 10.1371/journal.pgen.0030078
[25]
Sato T, Takahoko M, Okamoto H (2006) HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis 44: 136–142. doi: 10.1002/gene.20196
[26]
Higashijima S, Hotta Y, Okamoto H (2000) Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer. J Neurosci 20: 206–218.
[27]
Minoux M, Rijli FM (2010) Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development 137: 2605–2621. doi: 10.1242/dev.040048
[28]
Yan YL, Willoughby J, Liu D, Crump JG, Wilson C, et al. (2005) A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development. Development 132: 1069–1083. doi: 10.1242/dev.01674
[29]
Jin Y, Zhang W, Li Q (2009) Origins and evolution of ADAR-mediated RNA editing. IUBMB Life 61: 572–578. doi: 10.1002/iub.207
[30]
Nechiporuk A, Raible DW (2008) FGF-dependent mechanosensory organ patterning in zebrafish. Science 320: 1774–1777. doi: 10.1126/science.1156547
[31]
Seeburg PH, Single F, Kuner T, Higuchi M, Sprengel R (2001) Genetic manipulation of key determinants of ion flow in glutamate receptor channels in the mouse. Brain Res 907: 233–243. doi: 10.1016/s0006-8993(01)02445-3
[32]
Kwak S, Kawahara Y (2005) Deficient RNA editing of GluR2 and neuronal death in amyotropic lateral sclerosis. J Mol Med 83: 110–120. doi: 10.1007/s00109-004-0599-z
[33]
Tu CT, Yang TC, Huang HY, Tsai HJ (2012) Zebrafish arl6ip1 is required for neural crest development during embryogenesis. PloS one 7: e32899. doi: 10.1371/journal.pone.0032899
[34]
Morrison RS, Wenzel HJ, Kinoshita Y, Robbins CA, Donehower LA, et al. (1996) Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death. J Neurosci 16: 1337–1345.
[35]
Xiang H, Hochman DW, Saya H, Fujiwara T, Schwartzkroin PA, et al. (1996) Evidence for p53-mediated modulation of neuronal viability. J Neurosci 16: 6753–6765.
[36]
Horsch M, Seeburg PH, Adler T, Aguilar-Pimentel JA, Becker L, et al. Requirement of the RNA-editing enzyme ADAR2 for normal physiology in mice. J Biol Chem 286: 18614–18622. doi: 10.1074/jbc.m110.200881
[37]
Whitney NP, Peng H, Erdmann NB, Tian C, Monaghan DT, et al. (2008) Calcium-permeable AMPA receptors containing Q/R-unedited GluR2 direct human neural progenitor cell differentiation to neurons. Faseb J 22: 2888–2900. doi: 10.1096/fj.07-104661
Gill SS, Pulido OM (2001) Glutamate receptors in peripheral tissues: current knowledge, future research, and implications for toxicology. Toxicol Pathol 29: 208–223. doi: 10.1080/019262301317052486
[40]
Wienholds E, Koudijs MJ, van Eeden FJ, Cuppen E, Plasterk RH (2003) The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat Genet 35: 217–218. doi: 10.1038/ng1251
[41]
Ying SY, Lin SL (2005) MicroRNA: fine-tunes the function of genes in zebrafish. Biochem Biophys Res Commun 335: 1–4.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310. doi: 10.1002/aja.1002030302
[44]
Keegan LP, Leroy A, Sproul D, O'Connell MA (2004) Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes. Genome Biol 5: 209.
[45]
Thisse C, Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3: 59–69. doi: 10.1038/nprot.2007.514
[46]
Cole LK, Ross LS (2001) Apoptosis in the developing zebrafish embryo. Dev Biol 240: 123–142. doi: 10.1006/dbio.2001.0432
[47]
Barrallo-Gimeno A, Holzchuh J, Driever W, Knapik EW (2004) Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function. Development 131: 1463–1477. doi: 10.1242/dev.01033
[48]
Alexandre D, Ghysen A (1999) Somatotopy of the lateral line projection in larval zebrafish. Proc Natl Acad Sci USA 96: 7558–7562. doi: 10.1073/pnas.96.13.7558
[49]
Chen YH, Lu YF, Ko TY, Tsai MY, Lin CY, et al. (2009) Zebrafish cdx1b regulates differentiation of various intestinal cell lineages. Dev Dyn 238: 1021–1032. doi: 10.1002/dvdy.21908
