title: Studies of post-transcriptional gene regulation in rat liver and hepatoma cells
Creators: name: Jacobs, Howard Trevor
Publisher: name: University of Glasgow
Year: 1980

description: Studies of the composition of cellular RXA populations have indicated that very considerable shifts in phenotype, such as maturation along a given differentiation pathway, the induction or cessation of growth, the expression of specialised physiological functions, or carcinogenic transformation, are marked by essentially quantitative rather than qualitative changes in gene expression. Furthermore, these phenotypic alterations seem to be brought about by a complex interaction of regulatory events, operating at multiple sites in the pathway of mRXA Synthesis. The rates of transcription, post- transcriptional processing, nucleocytoplasmic transport, cytoplasmic mRNA turnover and recruitment into translationally active polysomes all appear to be subject to sequence-selective controls whose specificity varies according to the physiological context. In this study, the poly(A)[+] mRNA and hnRNA populations of rat liver and hepatoma (HTC) cells have been compaired by various hybridisation methodologies (single-copy DNA saturation, kinetics of cDNA cross-hybridisation, titration against cloned cDNAs) in order to characterise the extent to which post-transcriptional controls determine the overall pattern of relative mRNA abundances. The results indicate that the two cell-types transcribe essentially the same sequences, and express the same set of genes on their polysomes. However, their patterns of relative mRNA abundances are radically different. Liver expresses a set of superabundant, presumably differentiation-specific mRNAs, encoding both secretory and intracellular products, which are orders of magnitude rarer in HTC cells. No such dramatic difference in mRNA frequencies is evident in the opposing direction; however, a considerable number of mRNAs at intermediate abundance in liver are modestly increased in concentration in the hepatoma. The superabundant liver mRNAs which are depleted in HTC cells appear to be regulated mainly at a post-transcriptional level, since the disparity in their concentrations in hnRNA in the two cell-types is much, less striking (3-fold, on average, as compared vith 100-fold at the level of polysomal mRNA). Two other aspects of post-transcriptional selection in rat liver emerge from rhe results. A large proportion of rhe poly(a)-adjacent sequence complexity of hnRNA is unrepresenred on the polysomes. This may represent a qualitative control mechanism, defining the characxeristic expressed "gene set" of a committed cell, a control which seems to function unaltered in the hepatoma. Liver hnRXA also contains a population of relatively abundant poly(A)-adjacent sequences whose levels on the polysomes appear to be post-transcripticnally suppressed. In order to obtain more detailed information on the levels at which mRNA abundances are generated and modulated between cell-types and to gain insight into the mechanisms responsible, it is necessary to develop in vitro systems which mainxain the physiological specificity of gene regxilation in vivo. The clear-cut, post-rranscriptionally determined differ-ences in mRNA abundance, both within and between the cell-types considered here, provide a set of convenient and sensirive assays for the degree TO which post-transcriptional selectivity is preserved in isolated nuclei. A system was therefore developed, based on detergent-treated HTC cell nuclei, incubated in the presence of cytosol protein. The medium used for the incubations supported ATP- and cytosol-dependent transporr of prelabelled mRXA and rRXA and maintained nuclear integrity. The composition of the population of polyadenylated molecules transported from (unlabelled) nuclei was investigated by various hybridisation methodologies. Despite low levels of both non-specific leakage, and possible release of residual adherent RXA of cytoplasmic origin, the bulk of in vitro-transported poly(A)[+] RNA could not be explained as being due to eitlier of tliese artifacts. Its partem of relative abundances indicated that higlily sequence-selective processing and/or transport ineclianisms are able to operate in vitro. Liver cytosol was found to have no effect on xhe specificity of mRXA transport from isolated HTC cell nuclei, even when highly sensitive assays (borh heterogeneous and cloned cDXAs for post-transcripxionally regulate: messengers were used. This cannot be formally interpreted until the precise degree of physiological equivalence of the system is established which must await a more precise characterisation of the relative contributions of intranuclear and cytoplasmic events to polysomeil abundances in vivo, using cloned cDNAs. The results are discussed in terms of their implications for post-transcriptional gene control mechanisms, in relation to development, growth, differentiation and carcinogenesis.