Identification of a novel splice variant for mouse and human interleukin-5

Igor Shilovskiy; Sergei Andreev; Dmitriy Mazurov; Ekaterina Barvinskaia; Svetlana Bolotova; Alexander Nikolskii; Ilya Sergeev; Artem Maerle; Dmitrii Kudlay; Musa Khaitov

doi:10.1016/j.heliyon.2020.e03586

Identification of a novel splice variant for mouse and human interleukin-5

Heliyon. 2020 Mar 18;6(3):e03586. doi: 10.1016/j.heliyon.2020.e03586. eCollection 2020 Mar.

Authors

Affiliations

¹ Laboratory of Antiviral Immunity, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
² Laboratory of Peptide Immunogens, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
³ Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Vavilova Street 34/5, Moscow, 119334, Russia.
⁴ Laboratory of Immunochemistry, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
⁵ Laboratory of Human Histocompatibility Genetics, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
⁶ Laboratory of Personalized Medicine and Molecular Immunology, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.

Abstract

Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30-40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

Keywords: Alternative splicing; Biomolecules; Cytokine; Gene expression; Genetics; Immunology; Lymphocytes; Protein expression.