Shilovskiy Igor, Andreev Sergei, Mazurov Dmitriy, Barvinskaia Ekaterina, Bolotova Svetlana, Nikolskii Alexander, Sergeev Ilya, Maerle Artem, Kudlay Dmitrii, Khaitov Musa
Laboratory of Antiviral Immunity, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
Laboratory of Peptide Immunogens, National Research Center Institute of Immunology of Federal Medico-biological Agency, Kashirskoe shosse 24, Moscow, 115522, Russia.
Heliyon. 2020 Mar 18;6(3):e03586. doi: 10.1016/j.heliyon.2020.e03586. eCollection 2020 Mar.
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.
白细胞介素及其受体的表达常常受到可变剪接的调控。白细胞介素-5受体α链的可变异构体已得到充分研究；然而，迄今为止，尚未有关于白细胞介素-5功能性可变剪接变体的数据报道。在本研究中，我们描述了一种小鼠和人类白细胞介素-5的新型剪接变体。这种新形式是在分析用一对设计用于克隆全长小鼠白细胞介素-5开放阅读框（ORF）的引物从不同小鼠淋巴组织扩增的PCR产物时发现的。在从脾脏、胸腺、淋巴结和血液中分离的刀豆蛋白A刺激的淋巴细胞中，除了典型的全长mRNA外，还检测到了一种单一的短异构体小鼠白细胞介素-5。它比经典形式短30 - 40个核苷酸，且丰度较低。对另一种形式的小鼠白细胞介素-5进行序列分析发现，它缺少外显子2（δ2）。使用针对剪接特异性引物的逆转录-聚合酶链反应（RT-PCR），我们获得了δ2形式表达的额外证据。为了验证小鼠白细胞介素-5δ2转录本是否被翻译成蛋白质，将对应于小鼠白细胞介素-5全长和δ2形式的编码序列克隆到表达质粒中。转染到人293T细胞系后，我们发现小鼠白细胞介素-5的短形式蛋白在细胞中表达并分泌到上清液中，但水平低于检测到的小鼠白细胞介素-5全长异构体。荧光显微镜检查显示小鼠白细胞介素-5δ2部分易位到细胞质中，而小鼠白细胞介素-5主要位于内质网内。这可以解释为什么δ2蛋白表达水平降低。使用类似的一组实验方法，我们也获得了人白细胞介素-5 mRNA也有δ2剪接形式（人白细胞介素-5δ2）的证据。在从健康人或哮喘患者外周血中分离的植物血凝素（PHA）激活的单核细胞中，通过RT-PCR可以可靠地检测到它。总之，我们的结果表明，人和小鼠白细胞介素-5都有一种可变的mRNA剪接异构体，它缺失外显子2，但仍在蛋白质水平表达。然而，评估白细胞介素-5δ2的表达、调控、生物学功能和临床意义还需要更全面的研究。
