Lu Xinrong, Ferreira Paulo A
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Invest Ophthalmol Vis Sci. 2005 Jun;46(6):1882-90. doi: 10.1167/iovs.04-1286.
Mutations in RPGRIP1 cause Leber congenital amaurosis. The human and bovine RPGRIP1 undergo alternative splicing. A single murine rpgrip1 transcript has been reported, but distinct expression profiles of RPGRIP1 isoforms exist between species in the retina. To elucidate the heterogeneity of RPGRIP1 isoforms and the degree of functional redundancy among these, we extended the analysis of RPGRIP1 to the region between exons 12 and 14, which undergoes significant alternative splicing.
Identification of alternative splice transcripts of murine and human RPGRIP1 was performed by reverse transcription-polymerase chain reaction (RT-PCR). The murine rpgrip1 isoforms were analyzed by immunoblot and immunocytochemistry analysis of murine retinas and transient transfected cultured cells.
A novel murine-specific transcript, rpgrip1b was identified. It arises from the extension of exon 13, leading to the premature truncation of rpgrip1 and deletion of its C2 and RID domains. It is predominantly expressed in the retina, where it is more abundant than the transcript(s) encompassing the constitutive exons 12 to 14. Conversely, the human retina lacks rpgrip1b, and the constitutive transcript is the major isoform. The subcellular distribution of rpgrip1b is distinct from its larger isoform, rpgrip1. In the photoreceptor inner segments and cells expressing enhanced green fluorescent protein (EGFP)-rpgrip1b, rpgrip1b is dispersed as punctate foci throughout the perikarya, where it colocalizes with a subpopulation of lysosomes.
These data support the RPGR-independent function of the isotype- and species-specific rpgrip1b in lysosome-related processes. The results further strengthen the model of the selective participation of distinct RPGRIP1 isoforms in different subcellular processes and molecular pathogenesis of RPGRIP1-allied diseases.
RPGRIP1 基因突变会导致莱伯先天性黑蒙。人类和牛的 RPGRIP1 会发生可变剪接。据报道，小鼠只有一种 rpgrip1 转录本，但视网膜中 RPGRIP1 异构体的表达谱在不同物种间存在差异。为了阐明 RPGRIP1 异构体的异质性以及它们之间的功能冗余程度，我们将 RPGRIP1 的分析扩展到外显子 12 和 14 之间发生显著可变剪接的区域。
通过逆转录聚合酶链反应（RT-PCR）鉴定小鼠和人类 RPGRIP1 的可变剪接转录本。通过对小鼠视网膜和瞬时转染的培养细胞进行免疫印迹和免疫细胞化学分析，对小鼠 rpgrip1 异构体进行分析。
鉴定出一种新的小鼠特异性转录本 rpgrip1b。它由外显子 13 的延伸产生，导致 rpgrip1 过早截断并缺失其 C2 和 RID 结构域。它主要在视网膜中表达，在视网膜中的丰度高于包含组成型外显子 12 至 14 的转录本。相反，人类视网膜缺乏 rpgrip1b，组成型转录本是主要异构体。rpgrip1b 的亚细胞分布与其较大的异构体 rpgrip1 不同。在光感受器内段和表达增强型绿色荧光蛋白（EGFP）-rpgrip1b 的细胞中，rpgrip1b 以点状灶的形式分散在整个核周体中，与一部分溶酶体共定位。
这些数据支持了异构体特异性和物种特异性的 rpgrip1b 在溶酶体相关过程中不依赖 RPGR 的功能。结果进一步强化了不同 RPGRIP1 异构体选择性参与不同亚细胞过程以及 RPGRIP1 相关疾病分子发病机制的模型。
