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.
For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs.
Previously, we have shown that in murine myoblasts prosomes are constituents of the nuclear matrix; a major part of the latter was found to be RNase sensitive. Here, we further define the RNA-dependent matrix in avian erythroblastosis virus (AEV) transformed erythroid cells in relation to its structure, presence of specific RNA, prosomes and/or proteasomes. These cells transcribe but do not express globin genes prior to induction. Electron micrographs show little difference in matrices treated with DNase alone or with both, DNase and RNase. In situ hybridization with alpha globin riboprobes shows that this matrix includes globin transcripts. Of particular interest is that, apparently, a nearly 35 kb long globin full domain transcript (FDT), including genes, intergenic regions and a large upstream domain is a part of the RNA-dependent nuclear matrix. The 23K-type of prosomes, previously shown to be co-localized with globin transcripts in the nuclear RNA processing centers, were found all over the nuclear matrix. Other types of prosomes show different distributions in the intact cell but similar distribution patterns on the matrix. Globin transcripts and at least 80% of prosomes disappear from matrices upon RNase treatment. Interestingly, the 19S proteasome modulator complex is insensitive to RNase treatment. Only 20S prosomes but not 26S proteasomes are thus part of the RNA-dependent nuclear matrix. We suggest that giant pre-mRNA and FDTs in processing, aligning prosomes and other RNA-binding proteins are involved in the organization of the dynamic nuclear matrix. It is proposed that the putative function of RNA within the nuclear matrix and, thus, the nuclear dynamic architecture, might explain the giant size and complex organization of primary transcripts and their introns.
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