Studies of myc gene expression and a novel endoribonuclease activity

Abstract

The general aim of this work was to study the regulation of cellular oncogene expression, with emphasis on the c-myc gene, which is de-regulated in several human and animal tumors. The steady-state expression level of c-myc mRNA or protein was analyzed in panels of human Blymphoid cell lines with different growth and transformation properties, and the general observation was a co-variation between high c-myc expression and a more malignant phenotype of the cell lines.

The regulation of a c-myc-related gene, B-myc, was studied in murine cell lines of neuronal origin. B-myc mRNA was found to be co-regulated with c- and N-myc during normal growth as well as induced differentiation. Properties of a cloned B-myc cDNA expressed in human lymphoma cells were investigated and showed a mainly nuclear localisation of the protein. Like c-myc, expression of B-myc in a DNA-replication assay revealed a replication stimulating effect.

An important property of c-myc is the rapid regulation of expression, both positive and negative, in response to various environmental stimuli. In a model system for rapid c-myc downregulation, heat-shock, the influence of different regulatory sequences in the c-myc gene was studied by analysis of cell lines carrying various rearrangements of the c-myc gene. It was found that c-myc expression was downregulated after heat-shock irrespective of chromosomal localization, promoter utilization and structure of the 5' end of the gene. mRNA variants with normally long half- lives were also rapidly eliminated, indicating a post-transcriptional component in the mechanism of downregulation.

Given the short mRNA half-life of c-myc and the importance of this regulatory level for the expression of the gene, cell extracts were assayed for putative catalytic factors that would be able to recognize c-myc RNA sequences as targets for degradation. An in vitro endoribonuclease assay was used with cytoplasmic extracts from human cells. An activity capable of cleaving the 3' non-coding region of c-myc mRNA at several sites was identified and partially purified by ion exchange chromatography. Some cleavage sites were mapped and included the AUUUA sequence motif (cleaved at AUU * UA) that is present in multiple copies in many short-lived oncogene and lymphokine mRNA species. RNA species with four iterations of the AUUUA motif were found to be degraded more rapidly in the assay than RNA with one copy.

To study a possible evolutionary conservation, bacterial RNA species were tested with the human activity and found to be cleaved in a manner similar to RNase E from E.coli. The activity was therefore denoted "human RNase E-like activity". In E.coli, cleavage by RNase E is of major importance for mRNA degradation and the finding of a similar enzymatic activity in human cells indicates a strong conservation of components involved in mRNA decay.