Hong Dong-Hyun, Li Tiansen
Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye Ear Infirmary, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2002 Nov;43(11):3373-82.
To examine the mechanism underlying transcript heterogeneity in the gene for the retinitis pigmentosa GTPase regulator (RPGR).
Transcript heterogeneity was analyzed by reverse transcription-polymerase chain reactions (RT-PCR), rapid amplification of cDNA ends (RACE), and transient expression of minigene constructs. Protein variants were identified by immunoblot analysis and by immunocytochemistry.
RPGR transcripts terminated either uniformly at the end of exon 19, producing the constitutive transcript with few variants, or at variable sites downstream from exon 15. The latter transcripts resembled the previously described open reading frame (ORF)14/15 variant, but the ORF14/15 exon was not found in full length. Instead, various portions of a purine-rich region were removed as introns. Numerous splice site combinations were used, giving rise to innumerable variants. Analysis of the purine-rich region found multiple exonic splicing enhancers (ESEs) known to promote splicing through interaction with serine-arginine repeat (SR) proteins. Antibodies targeting different regions of RPGR detected a multitude of RPGR proteins in photoreceptors, concentrated in the connecting cilium. Predominant ORF14/15-encoded RPGR polypeptides migrated at approximately 200 kDa and were photoreceptor specific.
The exceptional heterogeneity in RPGR transcript processing results primarily from a novel form of alternative RNA splicing mediated by multiple exonic splicing enhancers. RPGR is composed of a population of proteins with a constant N-terminal core encompassing the RCC1 homology domain followed by a C-terminal portion of variable lengths and sequences.
研究视网膜色素变性GTP酶调节蛋白（RPGR）基因转录本异质性的潜在机制。
通过逆转录-聚合酶链反应（RT-PCR）、cDNA末端快速扩增（RACE）和小基因构建体的瞬时表达分析转录本异质性。通过免疫印迹分析和免疫细胞化学鉴定蛋白质变体。
RPGR转录本要么在外显子19末端统一终止，产生变体较少的组成型转录本，要么在外显子15下游的可变位点终止。后一种转录本类似于先前描述的开放阅读框（ORF）14/15变体，但未发现全长的ORF14/15外显子。相反，富含嘌呤区域的不同部分作为内含子被去除。使用了多种剪接位点组合，产生了无数变体。对富含嘌呤区域的分析发现了多个外显子剪接增强子（ESE），已知这些增强子通过与丝氨酸-精氨酸重复（SR）蛋白相互作用来促进剪接。靶向RPGR不同区域的抗体在光感受器中检测到大量RPGR蛋白，集中在连接纤毛中。主要的ORF14/15编码的RPGR多肽迁移约200 kDa，具有光感受器特异性。
RPGR转录本加工过程中异常的异质性主要源于由多个外显子剪接增强子介导的一种新型可变RNA剪接形式。RPGR由一群蛋白质组成，其N端核心恒定，包含RCC1同源结构域，随后是长度和序列可变的C端部分。
