Medical Research Building, Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton BN1 9PS, U.K.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Biochem J. 2018 Jun 29;475(12):2091-2105. doi: 10.1042/BCJ20170962.
DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.
DIS3(姐妹染色单体连接缺陷)是 exosome 的催化亚基,exosome 是一种参与 RNA 3'-5'降解的蛋白质复合物。DIS3 是一种高度保守的核酸外切酶,也称为 Rrp44。全球测序研究表明,DIS3 在多种癌症中发生突变,多发性骨髓瘤的发生率相当高。在这项工作中,我们鉴定了 DIS3 的两种蛋白编码同工型。这两种同工型都具有功能相关性,是由选择性剪接产生的。它们彼此之间的区别在于其 N 端 PIN(PilT N 端)结构域的大小不同,该结构域已被证明具有内切核酸酶活性,并将 DIS3 连接到 exosome。同工型 1 编码完整的 PIN 结构域,而同工型 2 的 PIN 结构域较短,并且缺失了具有保守氨基酸的片段。我们对全长 DIS3 的两种同工型和分离的 PIN 结构域进行了生化活性测定。我们发现,同工型 2 尽管缺失了部分 PIN 结构域,但比同工型 1 具有更强的内切核酸酶活性。对现有结构信息的检查使我们能够提出一个假设来解释这种改变的行为。我们的结果还表明,与同工型 2 相比,多发性骨髓瘤患者细胞和所有测试的癌细胞系中同工型 1 的水平更高,而急性髓细胞性白血病和慢性骨髓单核细胞性白血病患者细胞和健康供体样本中同工型 1 和 2 的水平相似。总之,我们的数据表明,两种同工型的比例发生显著变化可能是血液系统癌症的症状。