Kiri Arpna, Goldspink Geoffrey
Department of Anatomy and Developmental Biology, Royal Free Hospital and University College London, UK.
J Muscle Res Cell Motil. 2002;23(2):119-29. doi: 10.1023/a:1020211729728.
The RNA-protein interactions of the myosin heavy chain (MyHC) 3' untranslated regions (3'UTRs) were investigated using gel mobility shift assays. Marine skeletal myosin heavy chain mRNAs were amplified using reverse transcription coupled with the polymerase chain reaction (RT-PCR). Four cloned MyHC sequences were identified as slow type 1, fast 2a, fast 2b and fast 2x. The 3'UTRs of the four MyHC mRNAs were shown to interact with muscle protein in a tissue-specific manner as illustrated by gel retardation assays with protein extracts from various tissues. Competition assays indicate that this interaction is specific to the MyHC 3'UTR sequence. UV cross-linking suggests that several small proteins bind to the 3'UTR's. Peptide sequencing identified aldolase A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as MyHC 3'UTR RNA-binding proteins. The implications of these interactions and post-transcriptional regulation of the MyHC genes are discussed.
利用凝胶迁移率变动分析研究了肌球蛋白重链(MyHC)3'非翻译区(3'UTR)的RNA-蛋白质相互作用。采用逆转录结合聚合酶链反应(RT-PCR)扩增海洋骨骼肌肌球蛋白重链mRNA。鉴定出四个克隆的MyHC序列,分别为慢肌1型、快肌2a型、快肌2b型和快肌2x型。如用来自各种组织的蛋白质提取物进行凝胶阻滞分析所示,四个MyHC mRNA的3'UTR以组织特异性方式与肌肉蛋白相互作用。竞争分析表明这种相互作用对MyHC 3'UTR序列具有特异性。紫外线交联表明几种小蛋白质与3'UTR结合。肽测序鉴定醛缩酶A和甘油醛-3-磷酸脱氢酶(GAPDH)为MyHC 3'UTR RNA结合蛋白。讨论了这些相互作用的意义以及MyHC基因的转录后调控。