Dissecting Gene Expression by Hybridisation in situ (FISH) and Indirect Immuno Fluorescence

The expression of the α-globin gene domain was studied in the chicken erythroleucemic AEV cells by systematic Northern blotting and in situ hybridisation, using riboprobes of 15 fragments. A 33 kb-long Full Domain Transcript (FDT) runs through the entire globin domain including, from the 5' to 3' side, a putative LCR, an upstream silencer or terminator element, a CpG island acting as transcriptional modulator, the π-, αD- and αA-globin gene cluster, and the 3'-side enhancer and silencer elements. The precise function of the globin FDT is not clear as yet; the possibility is not excluded, however, that it may be the physical precursor of the individual globin pre-mRNAs. Most interestingly, we were able recently to give evidence for the first time, that the main function of high Mr RNAs of globin FDT-type may be at the level of the organisation of the nuclear matrix where they act as a structural backbone, assembling RNA-binding proteins.
Among the latter are the Prosomes (PS) which constitute a population of particles (Mr 720 kD) built of variable combinatories of 2 x 14 protein subunits. They were first observed as sub-complexes of cytoplasmic untranslated mRNPs, often associated to the cytoskeleton, as well as of nuclear pre-mRNPs. Some of their subunits have RNase activity. However, they constitute also the proteolytic core of the 26 S proteasomes. Acting on the biosynthetic as well as catabolic pathways they are involved, thus, in protein homeostasis. Studying the nuclear phase of the Prosomes on Rat Myoblasts we had identified them as components of the nuclear matrix, attached to chromatin, DNA and RNA; they where found to form specific but variable distribution patterns, lining up in particular around the nucleoli. On AEV cells, immune-fluorescence using monoclonal Abs specific to PS and the 19S Proteasome Regulator (ATPase) subunits Rpt3 (S6b, TBP7) and Rpt5 (S6a, TBP1), was combined with in situ hybridisation of globin riboprobes.
In transformed AEV cells, globin transcripts appear in the nuclear lumen and concentrate around the nucleoli. After induction of hemoglobin synthesis in these thermo-sensitive cells, transcripts move to two RNA processing centres (PCs) where partially processed transcripts accumulate. Interestingly, within the PCs, globin RNA co-localises with nuclear PS patches were, specifically, the p23K-type PS accumulate. Surprisingly, sites of coincidence of p23K-type PS and globin mRNA appear in the cytoplasm as well, at sites where the untranslated globin mRNPs concentrate. This indicates that globin transcripts might be transported on the nuclear matrix from the PCs to the nuclear periphery and into the cytoplasm along "Prosome trails".
Preparing nuclear matrices from AEV cells we found once more the 23K-type prosomes as constituents of the matrix networks lining up, as in myoblasts, around the nucleoli. Simultaneous hybridisation in situ with riboprobes for the globin genes and extra-genic FDT segments showed the presence of these transcripts in the matrix network as well. Treatment with RNase suppressed the globin signals completely and removed up to 90 % of the 23K prosomes. In contrast and most interestingly, the 19S regulator complex of the proteasome was not sensitive to RNase treatment indicating, that the 20S prosomes but not the 26S proteasomes are on the RNA matrix.
Carrying over the prosomes and other RNP proteins from the chromatin to the nuclear periphery during processing, pre-mRNA may preserve protein alignments assembled originally on the DNA-derived nuclear matrix. The integration of primary transcripts of the globin FDT-type allows suggesting, that they may represent the organisational principle of an RNA-dependent part of the nuclear matrix within which processing and mRNA transport proceed. Acting as a 3D-backbone for a dynamic part of the nuclear matrix, at instars of the precursors of ribosomal RNA (pre-rRNA) and ribosomes which form the nucleoli, the apparently useless length of primary transcripts and their introns finds a logical interpretation. Fragments of coding information would thus be inserted into supporting RNA forming 3D structures, as are in case of proteins the sites of catalytic or other activities, allowing specific processes to be controlled in space and time during gene expression.