Recessive Truncating Mutations in ALKBH8 Cause Intellectual Disability and Severe Impairment of Wobble Uridine Modification
2019; Elsevier BV; Volume: 104; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2019.03.026
ISSN1537-6605
AutoresDorota Monies, Cathrine Broberg Vågbø, Mohammad Al-Owain, Suzan Alhomaidi, Fowzan S. Alkuraya,
Tópico(s)Folate and B Vitamins Research
ResumoThe wobble hypothesis was proposed to explain the presence of fewer tRNAs than possible codons. The wobble nucleoside position in the anticodon stem-loop undergoes a number of modifications that help maintain the efficiency and fidelity of translation. AlkB homolog 8 (ALKBH8) is an atypical member of the highly conserved AlkB family of dioxygenases and is involved in the formation of mcm5s2U, (S)-mchm5U, (R)-mchm5U, mcm5U, and mcm5Um at the anticodon wobble uridines of specific tRNAs. In two multiplex consanguineous families, we identified two homozygous truncating ALKBH8 mutations causing intellectual disability. Analysis of tRNA derived from affected individuals showed the complete absence of these modifications, consistent with the presumptive loss of function of the variants. Our results highlight the sensitivity of the brain to impaired wobble modification and expand the list of intellectual-disability syndromes caused by mutations in genes related to tRNA modification. The wobble hypothesis was proposed to explain the presence of fewer tRNAs than possible codons. The wobble nucleoside position in the anticodon stem-loop undergoes a number of modifications that help maintain the efficiency and fidelity of translation. AlkB homolog 8 (ALKBH8) is an atypical member of the highly conserved AlkB family of dioxygenases and is involved in the formation of mcm5s2U, (S)-mchm5U, (R)-mchm5U, mcm5U, and mcm5Um at the anticodon wobble uridines of specific tRNAs. In two multiplex consanguineous families, we identified two homozygous truncating ALKBH8 mutations causing intellectual disability. Analysis of tRNA derived from affected individuals showed the complete absence of these modifications, consistent with the presumptive loss of function of the variants. Our results highlight the sensitivity of the brain to impaired wobble modification and expand the list of intellectual-disability syndromes caused by mutations in genes related to tRNA modification. The discovery of the genetic code and the mechanism by which tRNA performs its central role in protein translation presented an apparent conundrum in that there are more possible codon combinations than there are tRNAs with the corresponding cognate anticodons. The "wobble hypothesis" was proposed as a solution wherein non-standard (i.e., non-Crick and Watson) binding is possible between the first (thus referred to as the "wobble") nucleotide of the anticodon stem-loop of tRNA and the third nucleotide of the trinucleotide codon in mRNA.1Crick F.H. Codon—anticodon pairing: the wobble hypothesis.J. Mol. Biol. 1966; 19: 548-555Crossref PubMed Scopus (1297) Google Scholar Among the numerous modifications of tRNA, there are specific modifications of the wobble nucleotide, which are thought to be critical for faithful recognition of the cognate and non-cognate codons and, consequently, correct and efficient translation.2Agris P.F. Decoding the genome: A modified view.Nucleic Acids Res. 2004; 32: 223-238Crossref PubMed Scopus (269) Google Scholar, 3Sprinzl M. Vassilenko K.S. Compilation of tRNA sequences and sequences of tRNA genes.Nucleic Acids Res. 2005; 33: D139-D140Crossref PubMed Scopus (350) Google Scholar For example, wobble uridines in eukaryotic tRNAs normally harbor a 5-methoxycarbonylmethyl (mcm5) or a 5-carbamoylmethyl (ncm5) side chain, sometimes in combination with a 2-thio (s2) or ribose 2′-O-methyl group. These modifications modulate the tRNAs' decoding properties—ncm5U is specifically seen in tRNAs that decode "family codon boxes," i.e., groups of four codons that all encode the same amino acid, whereas tRNAs that harbor mcm5U decode "split codon boxes" that code for different amino acids by only varying the last purine or pyrimidine.2Agris P.F. Decoding the genome: A modified view.Nucleic Acids Res. 2004; 32: 223-238Crossref PubMed Scopus (269) Google Scholar Members of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase superfamily are dioxygenases that incorporate oxygen into their product in a reaction that converts O2 and 2OG to CO2 and succinate. These enzymes catalyze a multitude of cellular processes at the levels of DNA (e.g., DNA repair), RNA (e.g., hypoxia-induced transcriptional regulation), and protein (e.g., posttranslational modification), as well as the epigenome.4Loenarz C. Schofield C.J. Expanding chemical biology of 2-oxoglutarate oxygenases.Nat. Chem. Biol. 2008; 4: 152-156Crossref PubMed Scopus (392) Google Scholar, 5Tahiliani M. Koh K.P. Shen Y. Pastor W.A. Bandukwala H. Brudno Y. Agarwal S. Iyer L.M. Liu D.R. Aravind L. Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4228) Google Scholar, 6Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins.Nature. 2006; 439: 811-816Crossref PubMed Scopus (1602) Google Scholar AlkB homolog 8 (encoded by ALKBH8 [MIM: 613306]) is a member of the highly conserved AlkB family of dioxygenases defined by the presence of a characteristic 2OG-Fe(II) domain, also called an AlkB-like domain after its ortholog in E. coli, AlkB. AlkB removes alkylation damage from DNA and RNA as part of the adaptive stress response.7Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.Nature. 2002; 419: 174-178Crossref PubMed Scopus (632) Google Scholar, 8Aas P.A. Otterlei M. Falnes P.Ø. Vågbø C.B. Skorpen F. Akbari M. Sundheim O. Bjørås M. Slupphaug G. Seeberg E. Krokan H.E. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA.Nature. 2003; 421: 859-863Crossref PubMed Scopus (518) Google Scholar Unlike its eight mammalian paralogs (including the better-known FTO alpha-ketoglutarate-dependent dioxygenase, noted for its role in obesity pathogenesis9Frayling T.M. Timpson N.J. Weedon M.N. Zeggini E. Freathy R.M. Lindgren C.M. Perry J.R. Elliott K.S. Lango H. Rayner N.W. et al.A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.Science. 2007; 316: 889-894Crossref PubMed Scopus (3339) Google Scholar, 10Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Crossref PubMed Scopus (165) Google Scholar), ALKBH8 possesses additional methyltransferase (MT) and RNA recognition domains, the latter thought to confer specificity for targeting modified tRNAs.11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar, 12Pastore C. Topalidou I. Forouhar F. Yan A.C. Levy M. Hunt J.F. Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.J. Biol. Chem. 2012; 287: 2130-2143Crossref PubMed Scopus (54) Google Scholar Studies of mouse and human cells demonstrated that ALKBH8 is involved in the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylhydroxymethyluridine ([S]-mchm5U) and its diastereomer (R)-mchm5U, 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um) at the anticodon wobble uridines of specific tRNAs.13Fu D. Brophy J.A. Chan C.T. Atmore K.A. Begley U. Paules R.S. Dedon P.C. Begley T.J. Samson L.D. Human AlkB homolog ABH8 Is a tRNA methyltransferase required for wobble uridine modification and DNA damage survival.Mol. Cell. Biol. 2010; 30: 2449-2459Crossref PubMed Scopus (147) Google Scholar, 14Fu Y. Dai Q. Zhang W. Ren J. Pan T. He C. The AlkB domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA.Angew. Chem. Int. Ed. Engl. 2010; 49: 8885-8888Crossref PubMed Scopus (112) Google Scholar, 15Leihne V. Kirpekar F. Vågbø C.B. van den Born E. Krokan H.E. Grini P.E. Meza T.J. Falnes P.Ø. Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA.Nucleic Acids Res. 2011; 39: 7688-7701Crossref PubMed Scopus (40) Google Scholar More specifically, it has been shown that the MT domain of ALKBH8 provides the mcm5U precursor of mcm5s2U in tRNALys(UUU), tRNAGln(UUG), and tRNAGlu (UUC), and of mchm5U in tRNAGly (UCC) and tRNAArg (UCG), whereas its AlkB-like domain catalyzes the hydroxylation of wobble mcm5U to (S)-mchm5U in tRNAGly (UCC). A second unknown oxygenase is predicted to be responsible for the hydroxylation of mcm5U to (R)-mchm5U in tRNAArg (UCG) 16. In humans and mice, prior ALKBH8-dependent methylation to generate mcm5U is strictly required for 2′-thiolation to occur. In the absence of ALKBH8, mcm5s2U-tRNAs thus largely contain the precursor 5-carboxymethyluridine (cm5U) rather than the thiolated 5-carboxymethyl-2-thiouridine (cm5s2U).11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar Furthermore, ALKBH8 generates mcm5U at the wobble position of the specialized tRNASec (UGA), which is further 2′-O-methylated to mcm5Um in response to intracellular selenium levels (reviewed reviewed by Hatfield and Gladyshev16Hatfield D.L. Gladyshev V.N. How selenium has altered our understanding of the genetic code.Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (543) Google Scholar). In mammals, the lack of a specific anti-codon for selenocysteine is circumvented by this 2′-O-methylation of tRNASec (UGA) wobble mcm5U such that UGA is recoded from stop to selenocysteine.11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar, 17Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C.J. Hill K.E. Burk R.F. McCloskey J.A. Hatfield D.L. Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec).J. Biol. Chem. 1993; 268: 14215-14223Abstract Full Text PDF PubMed Google Scholar, 18Choi I.S. Diamond A.M. Crain P.F. Kolker J.D. McCloskey J.A. Hatfield D.L. Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes.Biochemistry. 1994; 33: 601-605Crossref PubMed Scopus (34) Google Scholar, 19Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.-A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. et al.Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar, 20Carlson B.A. Xu X.-M. Gladyshev V.N. Hatfield D.L. Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA.J. Biol. Chem. 2005; 280: 5542-5548Crossref PubMed Scopus (124) Google Scholar Like thiolation, 2′-O-methylation also requires prior methylation by ALKBH8.21Motorin Y. Helm M. RNA nucleotide methylation.Wiley Interdiscip. Rev. RNA. 2011; 2: 611-631Crossref PubMed Scopus (311) Google Scholar Thus, the ALKBH8 activity is likely to be critical for the 25 proteins that contain selenocysteine, including glutathione peroxidases (Gpx) and thioredoxin reductases (TrxRs), which are critical for the detoxification of reactive oxygen species (ROS).22Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar Indeed, when exposed to high ROS levels, mouse cells respond by increasing the levels of Alkbh8 and mcm5Um in order to increase the availability of Gpx1, Gpx3, Gpx4, Gpx6, and Txnrd1.23Endres L. Begley U. Clark R. Gu C. Dziergowska A. Małkiewicz A. Melendez J.A. Dedon P.C. Begley T.J. Alkbh8 regulates selenocysteine-protein expression to protect against reactive oxygen species damage.PLoS ONE. 2015; 10: e0131335Crossref PubMed Scopus (51) Google Scholar The yeast ortholog of ALKBH8 is Trm9, and its deficiency sensitizes yeast to DNA-damage-induced killing, most likely due to impaired translation of DNA-damage-response (DDR) proteins through impaired wobble uridine modification.24Begley U. Dyavaiah M. Patil A. Rooney J.P. DiRenzo D. Young C.M. Conklin D.S. Zitomer R.S. Begley T.J. Trm9-catalyzed tRNA modifications link translation to the DNA damage response.Mol. Cell. 2007; 28: 860-870Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Although several abnormalities have been observed in mouse embryonic fibroblasts (MEFs) derived from Alkbh8−/− mice (e.g., increased DNA damage at baseline and in response to DNA-damaging agents),23Endres L. Begley U. Clark R. Gu C. Dziergowska A. Małkiewicz A. Melendez J.A. Dedon P.C. Begley T.J. Alkbh8 regulates selenocysteine-protein expression to protect against reactive oxygen species damage.PLoS ONE. 2015; 10: e0131335Crossref PubMed Scopus (51) Google Scholar Alkbh8−/− mice are described as phenotypically normal.11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar However, these mice do recapitulate some of the MEF abnormalities, specifically the lack of mcm5U, mcm5s2U, and mcm5Um modifications, and the low levels of Gpx1.11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar Similarly, ALKBH8 knockdown in human cells leads to decreased mcm5U levels, and its expression is induced by DNA damage.13Fu D. Brophy J.A. Chan C.T. Atmore K.A. Begley U. Paules R.S. Dedon P.C. Begley T.J. Samson L.D. Human AlkB homolog ABH8 Is a tRNA methyltransferase required for wobble uridine modification and DNA damage survival.Mol. Cell. Biol. 2010; 30: 2449-2459Crossref PubMed Scopus (147) Google Scholar There are no diseases linked to ALKBH8 mutations, despite the growing list of Mendelian disorders caused by mutations in various components of tRNA modification machinery. One such component includes ADAT3 (encoded by ADAT3 [MIM: 615302]), which is required for the deamination of adenosine to inosine at the wobble position of tRNA that decodes "family codon boxes."25Alazami A.M. Hijazi H. Al-Dosari M.S. Shaheen R. Hashem A. Aldahmesh M.A. Mohamed J.Y. Kentab A. Salih M.A. Awaji A. et al.Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus.J. Med. Genet. 2013; 50: 425-430Crossref PubMed Scopus (75) Google Scholar On the basis of two independent biallelic mutational events in two multiplex families, we suggest in this study that the phenotype of ALKBH8 deficiency in humans is intellectual disability (ID). Furthermore, we show that ALKBH8 deficiency in these individuals is associated with absent (S)-mchm5U, (R)-mchm5U, mcm5Um, and mcm5s2U modifications in total tRNA. The index individual in family 1 (IV:13, Figure 1) is a 12-year old boy with ID, epilepsy, and history of global developmental delay (GDD). He had normal prenatal and birth history. Seizure onset was at age 1 year, and his seizures have been well controlled by medication. He sat at age 1 year and walked at age 2 years. Currently, he has very limited expressive language and an IQ (intelligence quotient) of 52. He is hyperactive with a very poor attention span. His complete lack of self-care and understanding of the concept of danger necessitates constant supervision. His medical history is largely unremarkable otherwise. His growth parameters are age appropriate, and he only has mild dysmorphism in the form of an overbite, a small penis, and undescended testicles. Echocardiogram was normal. Brain magnetic resonance imaging (MRI) revealed normal findings except for a well-defined rounded lesion seen in the right transverse sinuses; this lesion is iso-intense on T1 and hyper-intense on T2 and most likely represents an arachnoid granulation. He has two affected siblings, a 15-year-old brother (family 1_IV:12) with ID, epilepsy, and GDD, and a 5-year-old sister with ID and GDD but no epilepsy (family 1_IV:16). The index individual in family 2 (IV:12) is a 16-year-old boy with ID, epilepsy, GDD, and congenital heart disease (ventricular septal defect [VSD]). VSD was repaired at age 1 year and a follow-up echocardiogram showed a thickened tri-leaflet aortic valve with no stenosis but with mild regurgitation. Neuropsychological assessment (via the standard tool Beery-Buktenica Developmental Test of Visual-Motor Integration) at age 12 years showed an age equivalency of 66 months and a standard score of 62 (mild degree of cognitive delay) and severe linguistic impairment. Social skills were consistent with his mental age. Like the index individual in family 1, he was diagnosed with attention deficit hyperactivity disorder (ADHD) of the combined type. The first seizure episodes were noted at 9–12 months of age, and his seizures are currently under good control with topiramate. He had a myringotomy tube inserted bilaterally and an adeno-tonsillectomy at age 2 years as treatment for recurrent otitis media and persistent middle-ear effusion. Growth parameters showed macrocephaly (occipitofrontal circumference [OFC] 60 cm at 16 years of age) but normal height and weight. He had curly hair and large and deep-set eyes. He appeared active, had a happy demeanor, and had occasional stereotypic movements (lateral neck shaking). He was mildly hypotonic. An MRI of his brain was normal. His initial electroencephalogram (EEG) was mildly abnormal and showed generalized slowing and episodes of generalized delta discharges, which might indicate underlying cortical irritability. His chromosomal analysis and single-nucleotide polymorphism (SNP) array study were negative. His acylcarnitine profile, biotinidase enzymatic activity, urine organic acid analysis, and plasma very-long-chain-fatty-acid analysis were unremarkable. Family 2 has three other boys who are affected with same phenotype as the index individual and three children (two girls and one boy) who are unaffected (Figure 1). The first affected brother (family 2_IV:7) is now 28 years old and has severe ID, large ears, and a long face. He developed seizures at age 2 years, but they ceased by age 5 years. The second affected brother (family 2_IV:8) is now 26 years old and has a history of prune belly syndrome (severe hypoplasia of abdominal wall muscles as a result of chronically increased intra-abdominal pressure during fetal development, typically in association with urinary tract malformations) and underwent bilateral ureteric reimplantation and bilateral orchiopexy. He has severe ID and a long face and large ears. He developed seizures at around age 2 years, but these ceased at age 15 years. He has normal OFC. The third affected brother (family 2_IV:10) is a 24-year-old male who was also born with prune belly syndrome and was found to have left solitary kidney. His OFC at the age of 16 years was 55.5 cm (95th centile). He has severe ID and epilepsy, which is fairly controlled by multiple antiepileptic medications. He had a normal brain MRI. Table 1 summarizes the clinical features of all affected individuals.Table 1Summary of Clinical Features in the Affected IndividualsCase IDFamily 1_IV:12Family 1_IV:13Family 1_IV:16Family 2_IV:7Family 2_IV:8Family 2_IV:10Family 2_IV:12GendermalemalefemalemalemalemalemaleAge15 years12 years5 years28 years26 years24 years16 yearsIDPPPPPPPEpilepsyPPAPPPPGDDPPPPPPPMRI brainNAarachnoid granulationNANANANNOther featuresmild dysmorphism, ADHDmild dysmorphismprune belly syndromeprune belly syndrome, unilateral renal agenesis, macrocephalyADHD, CHD, macrocephalyAbbreviations are as follows: A, absent; ADHD, attention deficit hyperactivity disorder; CHD, congenital heart disease; N, normal; NA, not available; and P, present. Open table in a new tab Abbreviations are as follows: A, absent; ADHD, attention deficit hyperactivity disorder; CHD, congenital heart disease; N, normal; NA, not available; and P, present. Individuals were initially tested as part of a large clinical exome sequencing effort (manuscript under review). The index individual in each family independently underwent clinical exome sequencing that reported a different homozygous variant of unknown significance in ALKBH8: GenBank: NM_001301010.1, exon12, c.1660C>T (p.Arg554∗) in family 1 and GenBank: NM_001301010.1, exon12, c.1794delC (p.Trp599Glyfs∗19) in family 2 (Figure 2). Both families were subsequently recruited under an IRB-approved research protocol with informed consent (KFSHRC RAC# 2121053). Blood samples were collected from the affected and unaffected members of each nuclear family in tubes containing ethylenediaminetetraacetic acid (EDTA) and sodium heparin for DNA extraction and establishment of lymphoblastoid cell lines (LCL) for RNA extraction, respectively. Autozygome analysis using AutoSNPa was based on regions of homozygosity ≥2 Mb in length as surrogates of autozygosity. By defining the candidate autozygome as the autozygous intervals that are exclusively shared by the affected members, we identified a single candidate locus corresponding to chr11q22.3 (Figure 1). This was further corroborated by linkage analysis (EasyLINKAGE package was used under the default parameters) that confirmed linkage of both families to the same candidate autozygous interval with a LOD (logarithm of the odds) score of 6 (Figure 1). Segregation analysis confirmed strict segregation of the variants with the disease in an autosomal-recessive fashion in both families, as shown on the pedigrees (Figure 1). We analyzed the modification status of nucleosides from affected-individual- and control-derived tRNA by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as described before with the mass transitions listed in Table S1.26Brickner J.R. Soll J.M. Lombardi P.M. Vågbø C.B. Mudge M.C. Oyeniran C. Rabe R. Jackson J. Sullender M.E. Blazosky E. et al.A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.Nature. 2017; 551: 389-393Crossref PubMed Scopus (50) Google Scholar To address whether ALKBH8 deficiency had an effect on the modification status of wobble uridines, we extracted total tRNA from LCL derived from affected and unaffected individuals from Family 1 and Family 2, as well as from independent control subjects, using miRNeasy Mini Kit (QIAGEN). We analyzed the modification status of nucleosides from affected-individual- and control-derived tRNA by LC-MS/MS (as described previously) with the mass transitions listed in Table S1.26Brickner J.R. Soll J.M. Lombardi P.M. Vågbø C.B. Mudge M.C. Oyeniran C. Rabe R. Jackson J. Sullender M.E. Blazosky E. et al.A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.Nature. 2017; 551: 389-393Crossref PubMed Scopus (50) Google Scholar LC-MS/MS is arguably the modification analysis approach with the highest specificity; it identifies each nucleoside by its molecular mass and molecular fragmentation pattern upon gas-phase collisions of the nucleoside. The liquid chromatography (LC) retention time is decided by the nucleosides' physio-chemical properties, and this offers additional characteristics that are used for identification (see Table S1 for details). These several layers of specificity—molecular mass, molecular fragmentation pattern, and LC retention time—make it possible to identify specific nucleoside modifications with a high degree of certainty even with the extremely complex background of biological samples. LC-MS/MS revealed that the wobble modifications mcm5s2U, (R)-mchm5U and (S)-mchm5U, and mcm5Um were readily detected in total tRNA from all unaffected family members and independent controls but were completely absent from total tRNA from affected individuals (Figure 3). Similarly, mcm5U was detected in all controls of both families and was completely absent in all affected individuals of family 2 (Figure 3). However, in family 1, a low (2%) LC-MS/MS signal that resembled the characteristics of mcm5U was found in all affected individuals (Figure 4), so although the mcm5U levels were also dramatically decreased in family 2 affected individuals, we cannot exclude the possibility that very low levels of mcm5U may still be present. However, the ribose-methylated form mcm5Um was completely absent in all affected individuals from both families (Figures 3 and 4),indicating aberrant modification of the selenocysteine-specific tRNASec that is required for the efficient expression of certain stress-related selenoproteins.11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar, 17Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C.J. Hill K.E. Burk R.F. McCloskey J.A. Hatfield D.L. Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec).J. Biol. Chem. 1993; 268: 14215-14223Abstract Full Text PDF PubMed Google Scholar, 18Choi I.S. Diamond A.M. Crain P.F. Kolker J.D. McCloskey J.A. Hatfield D.L. Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes.Biochemistry. 1994; 33: 601-605Crossref PubMed Scopus (34) Google Scholar, 19Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.-A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. et al.Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (114) Google Scholar, 20Carlson B.A. Xu X.-M. Gladyshev V.N. Hatfield D.L. Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA.J. Biol. Chem. 2005; 280: 5542-5548Crossref PubMed Scopus (124) Google ScholarFigure 4ALKBH8 Mutations Are Associated with Abnormal Wobble Uridine ModificationShow full captionLC-MS/MS quantification of wobble uridine modifications and the independent tRNA modification m6A in total tRNA from patients (P, n = 7), unaffected family controls (UN, n = 6), and independent controls (IC, n = 3). Each dot in the bar graphs represents an individual from the indicated group. The levels are expressed as ratios of modified to unmodified nucleosides for all modifications except mcm5Um, where no standard was available. For mcm5Um, the levels are expressed as a ratio to the maximum recorded level in the sample set.View Large Image Figure ViewerDownload Hi-res image Download (PPT) LC-MS/MS quantification of wobble uridine modifications and the independent tRNA modification m6A in total tRNA from patients (P, n = 7), unaffected family controls (UN, n = 6), and independent controls (IC, n = 3). Each dot in the bar graphs represents an individual from the indicated group. The levels are expressed as ratios of modified to unmodified nucleosides for all modifications except mcm5Um, where no standard was available. For mcm5Um, the levels are expressed as a ratio to the maximum recorded level in the sample set. Thus, and in agreement with previous data on Alkbh8-deficient mice,11Songe-Møller L. van den Born E. Leihne V. Vågbø C.B. Kristoffersen T. Krokan H.E. Kirpekar F. Falnes P.Ø. Klungland A. Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.Mol. Cell. Biol. 2010; 30: 1814-1827Crossref PubMed Scopus (152) Google Scholar, 27van den Born E. Vågbø C.B. Songe-Møller L. Leihne V. Lien G.F. Leszczynska G. Malkiewicz A. Krokan H.E. Kirpekar F. Klungland A. Falnes P.Ø. ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.Nat. Commun. 2011; 2: 172Crossref PubMed Scopus (121) Google Scholar we detected substantial amounts of cm5U, the precursor of mcm5U, in total tRNA from affected individuals, whereas no cm5U was detected in total tRNA from controls (Figures 3
Referência(s)