Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, 15701 Athens, Greece.
Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, 15701 Athens, Greece.
Gene. 2019 May 30;699:135-144. doi: 10.1016/j.gene.2019.02.072. Epub 2019 Mar 5.
Next-generation sequencing (NGS) technology is highly expected to help researchers disclose the complexity of alternative splicing and understand its association with carcinogenesis. Alternative splicing alterations are firmly associated with multiple malignancies, in terms of functional roles in malignant transformation, motility, and/or metastasis of cancer cells. One perfect example illustrating the connection between alternative splicing and cancer is the human protein arginine methyltransferase 1 (PRMT1) gene, previously cloned from members of our research group and involved in a variety of processes including transcription, DNA repair, and signal transduction. Two splice variants of PRMT1 (variants v.1 and v.2) are downregulated in breast cancer. In addition, PRMT1 v.2 promotes the survival and invasiveness of breast cancer cells, while it could serve as a biomarker of unfavorable prognosis in colon cancer patients. The aim of this study was the molecular cloning of novel alternative splice variants of PRMT1 with the use of 3' RACE coupled with NGS technology. Extensive bioinformatics and computational analysis revealed a significant number of 19 novel alternative splicing events between annotated exons of PRMT1 as well as one novel exon, resulting in the discovery of multiple PRMT1 transcripts. In order to validate the full sequence of the novel transcripts, RT-PCR was carried out with the use of variant-specific primers. As a result, 58 novel PRMT1 transcripts were identified, 34 of which are mRNAs encoding new protein isoforms, whereas the rest 24 transcripts are candidates for nonsense-mediated mRNA decay (NMD).
下一代测序（NGS）技术有望帮助研究人员揭示可变剪接的复杂性，并了解其与致癌作用的关联。可变剪接的改变与多种恶性肿瘤密切相关，在癌细胞的恶性转化、运动和/或转移的功能作用方面。一个完美的例子说明了可变剪接与癌症之间的联系，即人类蛋白精氨酸甲基转移酶 1（PRMT1）基因，该基因最初是从我们研究小组的成员中克隆出来的，参与了包括转录、DNA 修复和信号转导在内的多种过程。PRMT1 的两个剪接变体（变体 v.1 和 v.2）在乳腺癌中下调。此外，PRMT1 v.2 促进乳腺癌细胞的存活和侵袭性，而它可以作为结肠癌患者预后不良的生物标志物。本研究的目的是使用 3' RACE 与 NGS 技术相结合，对 PRMT1 的新型可变剪接变体进行分子克隆。广泛的生物信息学和计算分析揭示了 PRMT1 注释外显子之间存在大量 19 种新型可变剪接事件以及一个新的外显子，从而发现了多个 PRMT1 转录本。为了验证新型转录本的全长序列，使用变体特异性引物进行了 RT-PCR。结果，鉴定出 58 种新型 PRMT1 转录本，其中 34 种是编码新蛋白异构体的 mRNA，而其余 24 种转录本是无意义介导的 mRNA 降解（NMD）的候选物。
