Molecular Biology Reports

, Volume 45, Issue 5, pp 699–711 | Cite as

Identification and expression of alternatively spliced novel isoforms of cancer associated MYD88 lacking death domain in mouse

  • Hassan Mubarak Ishqi
  • Mohammed Amir Husain
  • Sayeed Ur Rehman
  • Tarique Sarwar
  • Mohammad TabishEmail author
Original Article


MYD88 is an adaptor protein known to involve in activation of NF-κB through IL-1 receptor and TLR stimulation. It consists of N-terminal death domain and C-terminal Toll/IL-R homology domain that mediates its interaction with IL-1R associated kinase and IL-1R/TLR, respectively. MYD88 contributes to various types of carcinogenesis due to its involvement in oncogene induced inflammation. In the present study, we have recognized two new alternatively spliced variants of MyD88 gene in mouse using bioinformatics tools and molecular biology techniques in combination. The newly identified non-coding exon (NE-1) from 5′ upstream region alternatively splices with either exon E-2 or exon E-5 to produce two novel transcript variants MyD88N1 and MyD88N2 respectively. The transcript variant MyD88N1 was expressed in several tissues studied while the variant MyD88N2 was found to be expressed only in the brain. The analysis of the upstream region of novel exon by in silico approach revealed new promoter region PN, which possess potential signature sequences for diverse transcription factors, suggesting complex gene regulation. Studies of post translational modifications of conceptualized amino acid sequences of these isoforms revealed diversity in properties. Western blot analysis further confirmed the expression of protein isoform MYD88N1.


MyD88 gene Alternative splicing Differential expression MYD88 isoforms Transcriptional variants 



Authors are thankful to Department of Biotechnology (DBT), New Delhi, India, for generous funding to MT (Grant No. BT/PR5271/BID/7/395/2012) and for the award of Senior Research Fellowship to HMI by council of scientific and industrial research (CSIR) (Sanction No-09/112(0493)/2013-EMR-I). We are also thankful to the Department of Biochemistry A.M.U., Aligarh for providing us the necessary facilities. The funders had no role in the study design, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest in this work.

Ethical approval

Mice (A/J) were purchased from animal house facility of Jamia Hamdard University, New Delhi, India. To bred animal in house, the institutional animal care and use committee and guidelines of the committee were followed. Experimentations were permitted by Ministry of Environment and Forests, GOI, under registration no. 714/02/a/CPCSEA. It was approved by the Institutional Animal Ethic Committee (IAEC) of Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh, India. All surgeries were performed under chloroform anaesthesia and maximum efforts were made to minimize the sufferings.

Supplementary material

11033_2018_4209_MOESM1_ESM.tif (5.8 mb)
Supplementary Figure 1 Multiple sequence alignment (Clustal Omega) of amino acid sequence of published and new isoforms of MYD88 in mouse. The names of the isoforms are shown adjacent to the sequence. The alignment clearly depicts the marked difference at the N-termini of these isoforms. The MYD88N1 sequence starts from the methionine present in exon E-3 which is represented by downward red arrow. The translation initiation point in case of MYD88N2 is present in the exon E-5 shown with the downward green arrow. The asterisk (*) and dash (-) shows identical residues and no residues respectively (TIF 5974 KB)
11033_2018_4209_MOESM2_ESM.tif (3.7 mb)
Supplementary Figure 2 CpG Plot generated by EMBOSS Cpgplot tool used for CpG Island prediction. The predicted promoter region sequence was used for all the promoters. CpG Islands were present in the regions of promoter PC and PN with observed/expected ratio > 0.60 and G + C percentage > 60. However, the putative CpG Islands were found in PN only (TIF 3744 KB)


  1. 1.
    Lord KA, Liebermann BH, Liebermann DA (1990) Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 5:1095–1097PubMedGoogle Scholar
  2. 2.
    Bonnert TP, Garka KE, Parnet P, Sonoda G, Testa JR, Sims JE (1997) The cloning and characterization of human MyD88: a member of an IL-1 receptor related family. FEBS Lett 402:81–84CrossRefPubMedGoogle Scholar
  3. 3.
    Ohnishi H, Tochio H, Kato Z, Orii KE, Li A, Kimura T, Hiroaki H, Kondo N, Shirakawa M (2009) Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signalling. Proc Natl Acad Sci USA 106:10260–10265CrossRefPubMedGoogle Scholar
  4. 4.
    Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124:783–801CrossRefPubMedGoogle Scholar
  5. 5.
    Lin SC, Lo YC, Wu H (2010) Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465:885–890CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ali S, Huber M, Kollewe C, Bischoff SC, Falk W, Martin MU (2007) IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc Natl Acad Sci USA 104:18660–18665CrossRefPubMedGoogle Scholar
  7. 7.
    Motshwene PG, Moncrieffe MC, Grossmann JG, Kao C, Ayaluru M, Sandercock AM, Robinson CV, Latz E, Gay NJ (2009) An oligomeric signalling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4. J Biol Chem 284:25404–25411CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Janssens S, Beyaert R (2002) A universal role for MyD88 in TLR/IL-1R-mediated signalling. Trends Biochem Sci 27:474–482CrossRefPubMedGoogle Scholar
  9. 9.
    Lord KA, Abdollahi A, Hoffman-Liebermann B, Liebermann DA (1990) Dissection of the immediate early response of myeloid leukemia cells to terminal differentiation and growth inhibitory stimuli. Cell Growth Differ 1:637–645PubMedGoogle Scholar
  10. 10.
    von Bernuth H, Picard C, Puel A, Casanova JL (2012) Experimental and natural infections in MyD88- and IRAK-4-deficient mice and humans. Eur J Immunol 42:3126–3135CrossRefGoogle Scholar
  11. 11.
    Hardiman G, Rock FL, Balasubramanian S, Kastelein RA, Bazan JF (1996) Molecular characterization and modular analysis of human MyD88. Oncogene 13:2467–2475PubMedGoogle Scholar
  12. 12.
    Janssens S, Burns K, Tschopp J, Beyaert R (2002) Regulation of interleukin-1 and lipopolysaccharide-induced NF-kB activation by alternative splicing of MyD88. Curr Biol 12:467–471CrossRefPubMedGoogle Scholar
  13. 13.
    Garcia MA, Baraniak AP, Lasda EL (2004) Alternative splicing in disease and therapy. Nat Biotechnol 22:535–546CrossRefGoogle Scholar
  14. 14.
    Azim S, Banday AR, Tabish M (2012) Identification of alternatively spliced multiple transcripts of 5-hydroxytryptamine receptor in mouse. Brain Res Bull 87:250–258CrossRefPubMedGoogle Scholar
  15. 15.
    Ghigna C, De Toledo M, Bonomi S, Valacca C, Gallo S, Apicella M, Eperon I, Tazi J, Biamonti G (2010) Pro-metastatic splicing of Ron proto-oncogene mRNA can be reversed: therapeutic potential of bifunctional oligonucleotides and indole derivatives. RNA Biol 7:495–503CrossRefPubMedGoogle Scholar
  16. 16.
    Lu H, Lin L, Sato S, Xing Y, Lee CJ (2009) Predicting functional alternative splicing by measuring RNA selection pressure from multigenome alignments. PLoS Comput Biol 5:e1000608CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Banday AR, Azim S, Tabish M (2012) Identification and expression analysis of three novel splice variants of protein kinase A catalytic β subunit gene in the mouse using combinatorial in silico and molecular biology approaches. FEBS J 279:572–585CrossRefPubMedGoogle Scholar
  18. 18.
    Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6:386–398CrossRefPubMedGoogle Scholar
  19. 19.
    Rehman SU, Ishqi HM, Husain MA, Sarwar T, Tabish M (2016) A novel exon generates ubiquitously expressed alternatively spliced new transcript of mouse Abcc4 gene. Gene 594:131–137CrossRefPubMedGoogle Scholar
  20. 20.
    Ishqi HM, Rehman SU, Sarwar T, Husain MA, Tabish M (2016) Identification of differentially expressed three novel transcript variants of mouse ARNT gene. IUBMB Life 68:122–135CrossRefPubMedGoogle Scholar
  21. 21.
    Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:597–603CrossRefGoogle Scholar
  22. 22.
    Charpilloz C, Veuthey AL, Chopard B, Falcone JL (2014) Motifs tree: a new method for predicting post-translational modifications. Bioinformatics 30:1974–1982CrossRefPubMedGoogle Scholar
  23. 23.
    Tsunoda T, Takagi T (1999) Estimating transcription factor bind ability on DNA. Bioinformatics 15:622–630CrossRefPubMedGoogle Scholar
  24. 24.
    Messeguer X, Escudero R, Farre D, Nunez O, Martinez J, Alba MM (2002) PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics 18:333–334CrossRefPubMedGoogle Scholar
  25. 25.
    Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V, Sullivan M (2012) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res 40:D261–D270CrossRefGoogle Scholar
  26. 26.
    Perkins ND (2007) Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8:49–62CrossRefPubMedGoogle Scholar
  27. 27.
    Safe S, Abdelrahim M (2005) Sp transcription factor family and its role in cancer. Eur J Cancer 41:2438–2448CrossRefPubMedGoogle Scholar
  28. 28.
    Katze MG, He Y, Gale M Jr (2002) Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2:675–687CrossRefPubMedGoogle Scholar
  29. 29.
    Liberg D, Sigvardsson M, Akerblad P, Peter A (2002) The EBF/Olf/Collier family of transcription factors: regulators of differentiation in cells originating from all three embryonal germ layers. Mol Cell Biol 22:8389–8397CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Cross SH, Charlton JA, Nan X, Bird AP (1994) Purification of CpG islands using a methylated DNA binding column. Nat Genet 6:236–244CrossRefPubMedGoogle Scholar
  31. 31.
    Attwood JT, Yung RL, Richardson BC (2002) DNA methylation and the regulation of gene transcription. Cell Mol Life Sci 59:241–257CrossRefPubMedGoogle Scholar
  32. 32.
    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23:94–101CrossRefPubMedGoogle Scholar
  33. 33.
    Daniels MA, Hogquist KA, Jameson SC (2002) Sweet ‘n’ sour: the impact of differential glycosylation on T cell responses. Nat Immunol 3:903–910CrossRefPubMedGoogle Scholar
  34. 34.
    Jeong JW, Bae MK, Ahn MY, Kim SH, Sohn TK, Bae MH, Yoo MA, Song EJ, Lee KJ, Kim KW (2002) Regulation and destabilization of HIF-1 alpha by ARD1-mediated acetylation. Cell 111:709–720CrossRefPubMedGoogle Scholar
  35. 35.
    Jedrzejewski PT, Girod A, Tholey A, Konig N, Thullner S, Kinzel V, Bossemeyer D (1998) A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry. Protein Sci 7:457–469CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Frottin F, Martinez A, Peynot P, Mitra S, Holz RC, Giglione C, Meinnel T (2006) The proteomics of N-terminal methionine cleavage. Mol Cell Proteom 5:232336–232349CrossRefGoogle Scholar
  37. 37.
    Venables JP (2004) Aberrant and alternative splicing in cancer. Cancer Res 64:7647–7765CrossRefPubMedGoogle Scholar
  38. 38.
    Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T (2011) Epigenetics in alternative pre-mRNA splicing. Cell 144:16–26CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Busch A, Hertel KJ (2015) Splicing predictions reliably classify different types of alternative splicing. RNA 21:1–11CrossRefGoogle Scholar
  40. 40.
    Wang JQ, Jeelall YS, Ferguson LL, Horikawa K (2014) Toll-Like receptors and cancer: MYD88 mutation and inflammation. Front Immunol 5:367PubMedPubMedCentralGoogle Scholar
  41. 41.
    Rehman SU, Husain MA, Sarwar T, Ishqi HM, Tabish M (2015) Modulation of alternative splicing by anticancer drugs. Wiley Interdiscip Rev RNA 6:369–379CrossRefPubMedGoogle Scholar
  42. 42.
    Salcedo R, Cataisson C, Hasan U, Yuspa SH, Trinchieri G (2013) MyD88 and its divergent toll in carcinogenesis. Trends Immunol 34:379–389CrossRefPubMedGoogle Scholar
  43. 43.
    Janssens S, Burns K, Vercammen E, Tschopp J, Beyaert R (2003) MyD88S, a splice variant of MyD88, differentially modulates NF-kappaB- and AP-1-dependent gene expression. FEBS Lett 548:103–107CrossRefPubMedGoogle Scholar
  44. 44.
    Muzio M, Ni J, Feng P, Dixit VM (1997) IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signalling. Science 278:1612–1615CrossRefPubMedGoogle Scholar
  45. 45.
    Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z (1997) MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7:837–847CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of Life SciencesA.M. UniversityAligarhIndia
  2. 2.Department of BiosciencesJamia Millia IslamiaNew DelhiIndia
  3. 3.Deutsches Diabetes-ZentrumLeibniz-Zentrum für Diabetes-Forschung an der Heinrich-Heine-Universität DüsseldorfDüsseldorfGermany

Personalised recommendations