Razin Sergey V, Rynditch Alla, Borunova Victoria, Ioudinkova Elena, Smalko Victor, Scherrer Klaus
Institut J Monod, 2, Place Jussieu, 75251 Paris, Cedex 05, France.
J Cell Biochem. 2004 Jun 1;92(3):445-57. doi: 10.1002/jcb.20066.
Giant nuclear transcripts, and in particular the RNAs of the globin gene domains which are much larger than their canonical pre-mRNAs, have been an enigma for many years. We show here that in avian erythroblastosis virus (AEV)-transformed chicken erythroleukaemic cells, where globin gene expression is abortive, the whole domain of alpha-globin genes is transcribed for about 33 kb in the globin direction and that this RNA is part of the nuclear matrix. Northern blot hybridisation with strand-specific riboprobes, recognising genes and intergenic sequences, and RT-PCR with downstream primers, show that the continuous full domain transcript (FDT) starts in the vicinity of a putative LCR and includes all the genes as well as known regulatory sites, the replication origin, and the DNA loop anchorage region in the upstream area. Absent in chicken fibroblasts, the globin FDT overlaps the major part of the ggPRX housekeeping gene that is transcribed in the opposite direction. RT-PCR and in situ hybridisation with genic and extra-genic globin probes demonstrated that the globin FDT is a component of the nuclear matrix. We suggest that the globin FDTs keep the domain in an active state, and the globin RNAs on the processing pathway are a component of the nuclear matrix. They may take part in the dynamic nuclear architecture when productively processed, or turn over slowly when globins are not synthesised.
巨大的核转录本,尤其是珠蛋白基因结构域的RNA,其大小远大于其经典前体mRNA,多年来一直是个谜。我们在此表明,在禽成红细胞增多症病毒(AEV)转化的鸡红白血病细胞中,珠蛋白基因表达失败,α珠蛋白基因的整个结构域在珠蛋白方向转录约33 kb,并且该RNA是核基质的一部分。用识别基因和基因间序列的链特异性核糖探针进行Northern印迹杂交,以及用下游引物进行RT-PCR,结果表明连续的全结构域转录本(FDT)起始于一个假定的LCR附近,包括所有基因以及已知的调控位点、复制起点和上游区域的DNA环锚定区域。珠蛋白FDT在鸡成纤维细胞中不存在,它与以相反方向转录的管家基因ggPRX的大部分重叠。用基因和基因外珠蛋白探针进行RT-PCR和原位杂交表明,珠蛋白FDT是核基质的一个组成部分。我们认为,珠蛋白FDT使该结构域保持活跃状态,加工途径上的珠蛋白RNA是核基质的一个组成部分。它们在有效加工时可能参与动态核结构,或者在不合成珠蛋白时缓慢周转。