Skip to main content
SpringerLink
Log in

Characterization of human gene locus CYYR1: a complex multi-transcript system

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Cysteine/tyrosine-rich 1 (CYYR1) is a gene we previously identified on human chromosome 21 starting from an in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.3), where no coding region had previously been predicted. CYYR1 was initially characterized as a four-exon gene, whose brain-derived cDNA sequencing predicts a 154-amino acid product. In this study we provide, with in silico and in vitro analyses, the first detailed description of the human CYYR1 locus. The analysis of this locus revealed that it is composed of a multi-transcript system, which includes at least seven CYYR1 alternative spliced isoforms and a new CYYR1 antisense gene (named CYYR1-AS1). In particular, we cloned, for the first time, the following isoforms: CYYR1-1,2,3,4b and CYYR1-1,2,3b, which present a different 3′ transcribed region, with a consequent different carboxy-terminus of the predicted proteins; CYYR1-1,2,4 lacks exon 3; CYYR1-1,2,2bis,3,4 presents an additional exon between exon 2 and exon 3; CYYR1-1b,2,3,4 presents a different 5′ untranslated region when compared to CYYR1. The complexity of the locus is enriched by the presence of an antisense transcript. We have cloned a long transcript overlapping with CYYR1 as an antisense RNA, probably a non-coding RNA. Expression analysis performed in different normal tissues, tumour cell lines as well as in trisomy 21 and euploid fibroblasts has confirmed a quantitative and qualitative variability in the expression pattern of the multi-transcript locus, suggesting a possible role in complex diseases that should be further investigated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Vitale L, Casadei R, Canaider S, Lenzi L, Strippoli P, D’Addabbo P, Giannone S, Carinci P, Zannotti M (2002) Cysteine and tyrosine-rich 1 (CYYR1), a novel unpredicted gene on human chromosome 21 (21q21.2), encodes a cysteine and tyrosine-rich protein and defines a new family of highly conserved vertebrate-specific genes. Gene 290:141–151

    Article  CAS  PubMed  Google Scholar 

  2. Pereboev AV, Ahmed N, thi Man N, Morris GE (2001) Epitopes in the interacting regions of beta-dystroglycan (PPxY motif) and dystrophin (WW domain). Biochim Biophys Acta 1527:54–60

    Article  CAS  PubMed  Google Scholar 

  3. Ilsley JL, Sudol M, Winder SJ (2002) The WW domain: linking cell signalling to the membrane cytoskeleton. Cell Signal 14:183–189

    Article  CAS  PubMed  Google Scholar 

  4. Vitale L, Frabetti F, Huntsman SA, Canaider S, Casadei R, Lenzi L, Facchin F, Carinci P, Zannotti M, Coppola D, Strippoli P (2007) Sequence, “subtle” alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors. BMC Cancer 7:66

    Article  PubMed Central  PubMed  Google Scholar 

  5. Gray KA, Daugherty LC, Gordon SM, Seal RL, Wright MW, Bruford EA (2013) Genenames.org: the HGNC resources in 2013. Nucleic Acids Res 41:D545–D552. doi:10.1093/nar/gks1066

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Engels WR (1993) Contributing software to the internet: the Amplify program. Trends Biochem Sci 18:448–450

    Article  CAS  PubMed  Google Scholar 

  7. Boratyn GM, Schäffer AA, Agarwala R, Altschul SF, Lipman DJ, Madden TL (2012) Domain enhanced lookup time accelerated BLAST. Biol Direct 7:12. doi:10.1186/1745-6150-7-12

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Letunic I, Doerks T, Bork P (2012) SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res 40:D302–D305. doi:10.1093/nar/gkr931

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Pierleoni A, Indio V, Savojardo C, Fariselli P, Martelli PL, Casadio R (2011) MemPype: a pipeline for the annotation of eukaryotic membrane proteins. Nucleic Acids Res 39:W375–W380. doi:10.1093/nar/gkr282

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. doi:10.1373/clinchem.2008.112797

    Article  CAS  PubMed  Google Scholar 

  11. Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, Penning LC, Toegel S (2010) MIQE précis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 11:74. doi:10.1186/1471-2199-11-74

    Article  PubMed Central  PubMed  Google Scholar 

  12. Casadei R, Strippoli P, D’Addabbo P, Canaider S, Lenzi L, Vitale L, Giannone S, Frabetti F, Facchin F, Carinci P, Zannotti M (2003) mRNA 5′ region sequence incompleteness: a potential source of systematic errors in translation initiation codon assignment in human mRNAs. Gene 321:185–193

    Article  CAS  PubMed  Google Scholar 

  13. Casadei R, Piovesan A, Vitale L, Facchin F, Pelleri MC, Canaider S, Bianconi E, Frabetti F, Strippoli P (2012) Genome-scale analysis of human mRNA 5′ coding sequences based on expressed sequence tag (EST) database. Genomics 100:125–130. doi:10.1016/j.ygeno.2012.05.012

    Article  CAS  PubMed  Google Scholar 

  14. Kozak M (2002) Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1–34

    Article  CAS  PubMed  Google Scholar 

  15. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034

  16. Facchin F, Vitale L, Bianconi E, Piva F, Frabetti F, Strippoli P, Casadei R, Pelleri MC, Piovesan A, Canaider S (2011) Complexity of bidirectional transcription and alternative splicing at human RCAN3 locus. PLoS ONE 6:e24508. doi:10.1371/journal.pone.0024508

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Patterson D (2009) Molecular genetic analysis of Down syndrome. Hum Genet 126:195–214

    Article  CAS  PubMed  Google Scholar 

  18. Kim H, Klein R, Majewski J, Ott J (2004) Estimating rates of alternative splicing in mammals and invertebrates. Nat Genet 36:915–916

    Article  CAS  PubMed  Google Scholar 

  19. Keren H, Lev-Maor G, Ast G (2010) Alternative splicing and evolution: diversification, exon definition and function. Nat Rev Genet 11:345–355. doi:10.1038/nrg2776

    Article  CAS  PubMed  Google Scholar 

  20. Blencowe BJ (2012) An exon-centric perspective. Biochem Cell Biol 90:603–612. doi:10.1139/o2012-019

    Article  CAS  PubMed  Google Scholar 

  21. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476. doi:10.1038/nature07509

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Irimia M, Blencowe BJ (2012) Alternative splicing: decoding an expansive regulatory layer. Curr Opin Cell Biol 24:323–332. doi:10.1016/j.ceb.2012.03.005

    Article  CAS  PubMed  Google Scholar 

  23. Staub E (2012) An interferon response gene expression signature is activated in a subset of medulloblastomas. Transl Oncol 5(4):297–304

    Article  PubMed Central  PubMed  Google Scholar 

  24. Ibragimova I, Slifker MJ, Maradeo ME, Banumathy G, Dulaimi E, Uzzo RG, Cairns P (2013) Genome-wide promoter methylome of small renal masses. PLoS ONE 8(10):e77309. doi:10.1371/journal.pone.0077309

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Pei J, Grishin NV (2012) Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors. Cell Signal 24:758–769. doi:10.1016/j.cellsig.2011.11.011

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Shi S, Notenboom S, Dumont ME, Ballatori N (2010) Identification of human gene products containing Pro–Pro-x-Tyr (PY) motifs that enhance glutathione and endocytotic marker uptake in yeast. Cell Physiol Biochem 25:293–306. doi:10.1159/000276570

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Ingham RJ, Colwill K, Howard C, Dettwiler S, Lim CS, Yu J, Hersi K et al (2005) WW domains provide a platform for the assembly of multiprotein networks. Mol Cell Biol 25:7092–7106

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Stukenberg PT, Lustig KD, McGarry TJ, King RW, Kuang J, Kirschner MW (1997) Systematic identification of mitotic phosphoproteins. Curr Biol 7:338–348

    Article  CAS  PubMed  Google Scholar 

  29. Pierleoni A, Martelli PL, Casadio R (2011) MemLoci: predicting subcellular localization of membrane proteins in eukaryotes Bioinformatics 27:1224–1230. doi:10.1093/bioinformatics/btr108

  30. Green RE, Lewis BP, Hillman RT, Blanchette M, Lareau LF, Garnett AT, Rio DC, Brenner SE (2003) Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes. Bioinformatics 19(Suppl 1):i118–i121

    Article  PubMed  Google Scholar 

  31. Schweingruber C, Rufener SC, Zünd D, Yamashita A, Mühlemann O (2013) Nonsense-mediated mRNA decay—mechanisms of substrate mRNA recognition and degradation in mammalian cells. Biochim Biophys Acta 1829(6–7):612–623. doi:10.1016/j.bbagrm.2013.02.005

    Article  CAS  PubMed  Google Scholar 

  32. Casadei R, Pelleri MC, Vitale L, Facchin F, Lenzi L, Canaider S, Strippoli P, Frabetti F (2011) Identification of housekeeping genes suitable for gene expression analysis in the zebrafish. Gene Expr Patterns 11:271–276. doi:10.1016/j.gep.2011.01.003

    Article  CAS  PubMed  Google Scholar 

  33. Xu J, Srinivas BP, Tay SY, Mak A, Yu X, Lee SG, Yang H, Govindarajan KR, Leong B, Bourque G, Mathavan S, Roy S (2006) Genomewide expression profiling in the zebrafish embryo identifies target genes regulated by Hedgehog signaling during vertebrate development. Genetics 174:735–752

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S (2010) Smoothened as a new therapeutic target for human osteosarcoma. Mol Cancer 9:5. doi:10.1186/1476-4598-9-5

    Article  PubMed Central  PubMed  Google Scholar 

  35. Prandini P, Deutsch S, Lyle R, Gagnebin M, Delucinge Vivier C, Delorenzi M, Gehrig C, Descombes P, Sherman S, Dagna Bricarelli F, Baldo C, Novelli A, Dallapiccola B, Antonarakis SE (2007) Natural gene expression variation in Down syndrome modulates the outcome of gene-dosage imbalance. Am J Hum Genet 81:252–263

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Smit AF, Riggs AD (1996) Tiggers and DNA transposon fossils in the human genome. Proc Natl Acad Sci USA 93:1443–1448

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Paulis M, Moralli D, Bensi M, De Carli L, Raimondi E (2004) Isolation from the horse genome of a new DNA transposon belonging to the Tigger family. Mamm Genome 15:399–403

    Article  CAS  PubMed  Google Scholar 

  38. Gotea V, Makalowski W (2006) Do transposable elements really contribute to proteomes? Trends Genet 22:260–267

    Article  CAS  PubMed  Google Scholar 

  39. Schumann GG, Gogvadze EV, Osanai-Futahashi M, Kuroki A, Münk C, Fujiwara H, Ivics Z, Buzdin AA (2010) Unique functions of repetitive transcriptomes. Int Rev Cell Mol Biol 285:115–188. doi:10.1016/B978-0-12-381047-2.00003-7

    Article  CAS  PubMed  Google Scholar 

  40. Costa FF (2007) Non-coding RNAs: lost in translation? Gene 386:1–10

    Article  CAS  PubMed  Google Scholar 

  41. Wapinski O (2011) Chang HY (2011) Long noncoding RNAs and human disease. Trends Cell Biol 21:354–361. doi:10.1016/j.tcb.2011.04.001 (Erratum. In: Trends Cell Biol 21:561)

    Article  CAS  PubMed  Google Scholar 

  42. Lehner B, Williams G, Campbell RD, Sanderson CM (2002) Antisense transcripts in the human genome. Trends Genet 18:63–65

    Article  CAS  PubMed  Google Scholar 

  43. Lapidot M, Pilpel Y (2006) Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms. EMBO Rep 7:1216–1222

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Carninci P (2008) Non-coding RNA transcription: turning on neighbours. Nat Cell Biol 10:1023–1024. doi:10.1038/ncb0908-1023

    Article  CAS  PubMed  Google Scholar 

  45. Beiter T, Reich E, Williams RW, Simon P (2009) Antisense transcription: a critical look in both directions. Cell Mol Life Sci 66:94–112. doi:10.1007/s00018-008-8381-y

    Article  CAS  PubMed  Google Scholar 

  46. Ho MR, Tsai KW, Lin WC (2012) A unified framework of overlapping genes: towards the origination and endogenic regulation. Genomics 100:231–239. doi:10.1016/j.ygeno.2012.06.011

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by “Fondazione del Monte di Bologna e Ravenna” grant to FFr, and University of Bologna “RFO” grants to FFr and RC. Conceived and designed the experiments: RC, MCP and FFr. Performed the experiments: RC, MCP, LV, MV, EM and FFr. Analysed the data: RC, MCP, LV, FFa, SC, PS, AP, EB, FP and FFr. Wrote the paper: RC, MCP and FFr. Participated to final revision and approval of paper: RC, MCP, LV, FFa, SC, PS, MV, AP, EB, EM, FP and FFr. The Authors are grateful to Gabriella Mattei and Michela Bonaguro for their excellent technical assistance with automated sequencing. The Authors wish to thank Dr. Paolo Comeglio for helping with the English revision of the manuscript.

Author information

Authors and Affiliations

  1. Department for Life Quality Studies, University of Bologna, C.so d’Augusto 237, 47921, Rimini, RN, Italy

    Raffaella Casadei & Elisa Mariani

  2. Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Unit of Histology, Embryology and Applied Biology, University of Bologna, Via Belmeloro, 8, 40126, Bologna, BO, Italy

    Maria Chiara Pelleri, Lorenza Vitale, Federica Facchin, Silvia Canaider, Pierluigi Strippoli, Matteo Vian, Allison Piovesan, Eva Bianconi & Flavia Frabetti

  3. Odontostomatological and Specialized Clinical Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131, Ancona, AN, Italy

    Francesco Piva

Authors
  1. Raffaella Casadei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Chiara Pelleri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lorenza Vitale
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Federica Facchin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Silvia Canaider
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pierluigi Strippoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matteo Vian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Allison Piovesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eva Bianconi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Elisa Mariani
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Francesco Piva
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Flavia Frabetti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Flavia Frabetti.

Additional information

Raffaella Casadei and Maria Chiara Pelleri have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11033_2014_3480_MOESM1_ESM.pdf

Online Resource 1 Primers and PCR conditions (Ta and cycles) used for CYYR1 locus transcripts RT-PCR (a) and for CYYR1-1,2,3,4 isoform quantitative expression real-time PCR analysis (b) § Gene region where we designed the specific primer. Exon (E), untraslated region (UTR) * PCR conditions indicated are annealing temperature (Ta) used in RT-PCR or real-time and number of cycles (Cy) (PDF 114 kb)

11033_2014_3480_MOESM2_ESM.pdf

Online Resource 2 a Schematic representation of the CYYR1-AS1 transcript. The arrows represent forward and reverse primers used to clone it (see Online Resource 1); the overlapping lines represent the amplification products (see Online Resource 4a for product size). b 1.5 % agarose gel loaded with human brain tissue RT-PCR products (5 µL) obtained using overlapping primers pairs for CYYR1-AS1 cloning. Amplicons (a) are labelled from 1 to 5 like the corresponding lane on agarose gel (b); M: 1 µL (500 ng) GeneRuler DNA Ladder Mix (PDF 108 kb)

11033_2014_3480_MOESM3_ESM.pdf

Online Resource 3 RT-PCR qualitative expression analysis of CYYR1 alternative splicing isoforms and antisense transcript in 19 normal human tissues. 1.5 % agarose gel loaded with human normal tissues RT-PCR products (5 μl). Unless otherwise specified, cycles of amplification were 35cy (standard conditions). Lanes: 1, stomach; 2, spleen; 3, colon; 4, lung; 5, liver; 6, pancreas; 7, adrenal gland; 8, peripheral blood leukocytes; 9, bone marrow; 10, thymus; 11, mammary gland; 12, skeletal muscle; 13, testis; 14, heart; 15, small intestine; 16, prostate; 17, brain; 18, placenta; 19, ovary; 20, RT negative control; 21, PCR negative control; 22, PCR positive control. M1: 1 μL (500 ng) size marker GeneRuler 1 kb DNA Ladder; M2: 1 μL (250 ng) size marker MBI 5-pBR322DNA/BsuRI; M3: 1 μL (500 ng) size marker GeneRuler (PDF 640 kb)

11033_2014_3480_MOESM4_ESM.pdf

Online Resource 4 RT-PCR results of the CYYR1 splicing isoforms and antisense transcript in tumour (a) and fibroblastic cell lines (b). 1.5 % agarose gel loaded with cell lines RT-PCR products (5 μL). Primer pairs used are listed in Online Resource 1a. M: 1 µL (500 ng) GeneRuler DNA Ladder Mix (in brackets from bottom to top 100, 500 and 1.000 bp). Lane 1: CYYR1-1,2,3,4b (primers #1 and #4); lane 2: CYYR1-1,2,3b (primers #1 and #9); lane 3: CYYR1-1b,2,3,4 (primers #12 and #2); lane 4: CYYR1-1,2,4 (primers #14 and #2); lane 5: CYYR1-1,2,2bis,3,4 (primers #5 and #15); lane 6: CYYR1-AS1 (primers #26 and #21) (PDF 1128 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Casadei, R., Pelleri, M.C., Vitale, L. et al. Characterization of human gene locus CYYR1: a complex multi-transcript system. Mol Biol Rep 41, 6025–6038 (2014). https://doi.org/10.1007/s11033-014-3480-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-014-3480-3

Keywords

Search

Navigation