A novel missense mutation of RPGR identified from retinitis pigmentosa affects splicing of the ORF15 region and causes loss of transcript heterogeneity
A novel missense mutation of RPGR identified from retinitis pigmentosa affects splicing of the ORF15 region and causes loss of transcript heterogeneity
复制标题
从色素性视网膜炎中鉴定出的 RPGR 的新错义突变影响 ORF15 区域的剪接并导致转录异质性丧失
DOI：
10.1016/j.bbrc.2020.06.109
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发表时间：
2020-10-15
影响因子：
3.1
通讯作者：
Chen, Jian-Huan
中科院分区：
文献类型：
--
作者：
Liu, Yan-Shan;Pan, Jia-Qi;Chen, Jian-Huan
色素性视网膜炎 GTP 酶调节器 (RPGR) 基因的突变是 X 连锁色素性视网膜炎 (RP) 的主要原因，其中 RPGR 的外显子开放阅读框 15 (ORF15) 发挥了重要作用。我们从 RP 的全外显子组测序中鉴定出 RPGR 的一个新的半合子错义突变 E585K。通过RNA-Seq分析和功能研究来研究该突变的潜在致病机制。我们的结果表明，该突变实际上影响了 RPGR ORF15 剪接。对人类视网膜的 RNA-Seq 分析以及随后在细胞中的验证揭示了 ORF15 区域中 RPGR 外显子 14 的 3'' 边界附近的复杂剪接模式，这是由各种选择性剪接事件 (ASE) 产生的。在人 293T 细胞中表达的野生型 RPGR 小基因在体外证实了这些 ASE。相比之下，如果没有检测到新的RNA种类，突变的小基因就会破坏ORF15区域的剪接模式，并导致RPGR转录本异质性的丧失。源自突变小基因的 RNA 种类主要是由外显子 14 中上游选择性 5' 剪接位点产生的较小的框架外转录本，这种转录本也在野生型 RPGR 中观察到。因此，我们的研究结果提供了关于 RPGR 外显子突变对 ORF15 区域选择性剪接的影响以及 RP 的潜在分子机制的见解。 (C) 2020 Elsevier Inc. 保留所有权利。
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP), in which exon open reading frame 15 (ORF15) of RPGR has been implicated to play a substantial role. We identified a novel hemizygous missense mutation E585K of RPGR from wholeexome sequencing of RP. RNA-Seq analysis and functional study were conducted to investigate the underlying pathogenic mechanism of the mutation. Our results showed that the mutation actually affected RPGR ORF15 splicing. RNA-Seq analysis of the human retina followed by validation in cells revealed a complex splicing pattern near the 3 '' boundary of RPGR exon 14 in the ORF15 region, resulting from a variety of alternative splicing events (ASEs). The wildtype RPGR mini-gene expressed in human 293T cells confirmed these ASEs in vitro. In contrast, without new RNA species detected, the mutant mini-gene disrupted the splicing pattern of the ORF15 region, and caused loss of RPGR transcript heterogeneity. The RNA species derived from the mutant mini-gene were predominated by a minor out-of-frame transcript that was also observed in wildtype RPGR, resulting from an upstream alternative 5 ' splice site in exon 14. Our findings therefore provide insights into the influence of RPGR exonic mutations on alternative splicing of the ORF15 region, and the underlying molecular mechanism of RP. (C) 2020 Elsevier Inc. All rights reserved.