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.
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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.
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Raffaella Casadei and Maria Chiara Pelleri have contributed equally to this work.
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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)
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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
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DOI: https://doi.org/10.1007/s11033-014-3480-3