Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform

Autoři: Natalia Berges aff001;  Meh Sameen Nawaz aff001;  Tuva Børresdatter Dahl aff001;  Lars Hagen aff003;  Magnar Bjørås aff001;  Jon K. Laerdahl aff001;  Ingrun Alseth aff001
Působiště autorů: Department of Microbiology, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway aff001;  Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet and Institute of Clinical Medicine, University of Oslo, Oslo, Norway aff002;  Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway aff003;  PROMEC Core Facility for Proteomics and Modomics, Norwegian University of Science and Technology and Central Norway Regional Health Authority, Trondheim, Norway aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225081


Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3’-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform.

Klíčová slova:

293T cells – Immunoprecipitation – Macrophages – Messenger RNA – Polymerase chain reaction – Recombinant proteins – Inosine – Ribozymes


1. Dalhus B, Arvai AS, Rosnes I, Olsen OE, Backe PH, Alseth I, et al. (2009) Structures of endonuclease V with DNA reveal initiation of deaminated adenine repair. Nat Struct Mol Biol 16: 138–143. doi: 10.1038/nsmb.1538 19136958

2. Feng H, Dong L, Klutz AM, Aghaebrahim N, Cao W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3'-exonuclease activity in endonuclease V. Biochemistry 44: 11486–11495. doi: 10.1021/bi050837c 16114885

3. Guo G, Ding Y, Weiss B. (1997) nfi, the gene for endonuclease V in Escherichia coli K-12. JBacteriol 179: 310–316.

4. Yao M, Hatahet Z, Melamede RJ, Kow YW. (1994) Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli. J Biol Chem 269: 16260–16268. 8206931

5. Lindahl T. (1993) Instability and decay of the primary structure of DNA. Nature 362: 709–715. doi: 10.1038/362709a0 8469282

6. Mannion N, Arieti F, Gallo A, Keegan LP, O’Connell MA. (2015) New insights into the biological role of mammalian ADARs; the RNA editing proteins. Biomolecules 5: 2338–2362. doi: 10.3390/biom5042338 26437436

7. Yasui M, Suenaga E, Koyama N, Masutani C, Hanaoka F, Gruz P, et al. (2008) Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA adduct, during translesion synthesis catalyzed by human DNA polymerases. J Mol Biol 377: 1015–1023. doi: 10.1016/j.jmb.2008.01.033 18304575

8. Alseth I, Dalhus B, Bjoras M. (2014) Inosine in DNA and RNA. Curr Opin Genet Dev 26: 116–123. doi: 10.1016/j.gde.2014.07.008 25173738

9. Dixit S, Henderson JC, Alfonzo JD. (2019) Multi-Substrate specificity and the evolutionary basis for interdependence in tRNA editing and methylation enzymes. Front Genet 10: 104. doi: 10.3389/fgene.2019.00104 30838029

10. Nawaz MS, Vik ES, Ronander ME, Solvoll AM, Blicher P, Bjørås M, et al. (2016) Crystal structure and MD simulation of mouse EndoV reveal wedge motif plasticity in this inosine-specific endonuclease. Sci Rep 6: 24979. doi: 10.1038/srep24979 27108838

11. Morita Y, Shibutani T, Nakanishi N, Nishikura K, Iwai S, Kuraoka I. (2013) Human endonuclease V is a ribonuclease specific for inosine-containing RNA. Nat Commun 4: 2273. doi: 10.1038/ncomms3273 23912718

12. Vik ES, Nawaz MS, Strom AP, Fladeby C, Bjoras M, Dalhus B, et al. (2013) Endonuclease V cleaves at inosines in RNA. NatCommun 4: 2271.

13. Nawaz MS, Vik ES, Berges N, Fladeby C, Bjoras M, Dalhus B, et al. (2016) Regulation of human endonuclease V activity and relocalization to cytoplasmic stress granules. J Biol Chem 291: 21786–21801. doi: 10.1074/jbc.M116.730911 27573237

14. Fladeby C, Vik ES, Laerdahl JK, Gran NC, Heggelund JE, Thorgaard E, et al. (2012) The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures. PLoS One 7: e47466. doi: 10.1371/journal.pone.0047466 23139746

15. Schouten KA, Weiss B. (1999) Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid. Mutat Res 435: 245–254. doi: 10.1016/s0921-8777(99)00049-x 10606815

16. Garcia-Caballero D, Perez-Moreno G, Estevez AM, Ruiz-Perez LM, Vidal AE, González-Pacanowska D. (2017) Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei. Sci Rep 7: 8505. doi: 10.1038/s41598-017-08910-1 28819113

17. Lee Y, Rio DC. (2015) Mechanisms and regulation of alternative pre-mRNA splicing. Annu Rev Biochem 84: 291–323. doi: 10.1146/annurev-biochem-060614-034316 25784052

18. Stastna M, Van Eyk JE. (2012) Analysis of protein isoforms: can we do it better? Proteomics 12: 2937–2948. doi: 10.1002/pmic.201200161 22888084

19. Maglott D, Ostell J, Pruitt KD, Tatusova T. (2011) Entrez Gene: gene-centered information at NCBI. Nucleic Acids Res 39: D52–57. doi: 10.1093/nar/gkq1237 21115458

20. O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, et al. (2016) Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 44: D733–745. doi: 10.1093/nar/gkv1189 26553804

21. Cunningham F, Achuthan P, Akanni W, Allen J, Amode MR, Armean IM, et al. (2019) Ensembl 2019. Nucleic Acids Res 47: D745–D751. doi: 10.1093/nar/gky1113 30407521

22. UniProt C (2019) UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res 47: D506–D515. doi: 10.1093/nar/gky1049 30395287

23. Jones DT, Cozzetto D. (2015) DISOPRED3: precise disordered region predictions with annotated protein-binding activity. Bioinformatics 31: 857–863. doi: 10.1093/bioinformatics/btu744 25391399

24. Duale N, Lindeman B, Komada M, Olsen AK, Andreassen A, Soderlund EJ, et al. (2007) Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles. Mol Cancer 6: 53. doi: 10.1186/1476-4598-6-53 17711579

25. Bronson DL, Andrews PW, Solter D, Cervenka J, Lange PH, Fraley EE. (1980) Cell line derived from a metastasis of a human testicular germ cell tumor. Cancer Res 40: 2500–2506. 7388807

26. Rappsilber J, Mann M, Ishihama Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2: 1896–1906. doi: 10.1038/nprot.2007.261 17703201

27. Pettersen HS, Galashevskaya A, Doseth B, Sousa MM, Sarno A, Visnes T, et al. (2015) AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature. DNA Repair (Amst) 25: 60–71.

28. Kim JI, Tohashi K, Iwai S, Kuraoka I. (2016) Inosine-specific ribonuclease activity of natural variants of human endonuclease V. FEBS Lett 590: 4354–4360. doi: 10.1002/1873-3468.12470 27800608

29. Zhang Z, Hao Z, Wang Z, Li Q, Xie W. (2014) Structure of human endonuclease V as an inosine-specific ribonuclease. Acta Crystallogr D Biol Crystallogr 70: 2286–2294. doi: 10.1107/S139900471401356X 25195743

30. Rodriguez JM, Rodriguez-Rivas J, Di Domenico T, Vazquez J, Valencia A, Tress ML. (2018) APPRIS 2017: principal isoforms for multiple gene sets. Nucleic Acids Res 46: D213–D217. doi: 10.1093/nar/gkx997 29069475

31. Mele M, Ferreira PG, Reverter F, DeLuca DS, Monlong J, Sammeth M, et al. (2015) Human genomics. The human transcriptome across tissues and individuals. Science 348: 660–665. doi: 10.1126/science.aaa0355 25954002

32. Persson H, Kvist A, Rego N, Staaf J, Vallon-Christersson J, Luts L, et al. (2011) Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene. Cancer Res 71: 78–86. doi: 10.1158/0008-5472.CAN-10-1869 21199797

33. Dedon PC, Tannenbaum SR. (2004) Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophys 423: 12–22. doi: 10.1016/ 14989259

34. Martin RM, Ter-Avetisyan G, Herce HD, Ludwig AK, Lattig-Tunnemann G, Cardoso MC. (2015) Principles of protein targeting to the nucleolus. Nucleus 6: 314–325. doi: 10.1080/19491034.2015.1079680 26280391

35. Dang CV, Lee WM. (1989) Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J Biol Chem 264: 18019–18023. 2553699

36. Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. (2010) The nucleolus under stress. Mol Cell 40: 216–227. doi: 10.1016/j.molcel.2010.09.024 20965417

37. Mi R, Ford-Zappala M, Kow YW, Cunningham RP, Cao W. (2012) Human endonuclease V as a repair enzyme for DNA deamination. Mutat Res 735: 12–18. doi: 10.1016/j.mrfmmm.2012.05.003 22664237

38. Rath M, Muller I, Kropf P, Closs EI, Munder M. (2014) Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front Immunol 5: 532. doi: 10.3389/fimmu.2014.00532 25386178

39. Patterson JB, Samuel CE. (1995) Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase. Mol Cell Biol 15: 5376–5388. doi: 10.1128/mcb.15.10.5376 7565688

Článek vyšel v časopise


2019 Číslo 11