Studies of myc gene expression and a novel endoribonuclease activity

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><h3>List of scientific papers</h3><p>I. Wennborg A, Aman P, Saranath D, Pear W, Sumegi J, Klein G (1987). Conversion of the lymphoma line "BJAB" by Epstein-Barr virus into phenotypically altered sublines is accompanied by increased c-myc mRNA levels. Int J Cancer. 40(2): 202-6. <br><a href="https://pubmed.ncbi.nlm.nih.gov/3038758">https://pubmed.ncbi.nlm.nih.gov/3038758</a><br><br></p><p>II. Wennborg AD, Altiok E, Moore JP, Ernberg I, Klein G (1991). Differential c-myc protein expression in Burkitts lymphomas and EBV-transformed lymphoblastoid lines. Eur J Cancer. 27(12): 1643-5. <br><a href="https://pubmed.ncbi.nlm.nih.gov/1782076">https://pubmed.ncbi.nlm.nih.gov/1782076</a><br><br></p><p>III. Asker C, Wennborg A, Steinitz M, Sugiaman D, Klein G (1995). Expression of b-myc in tumor cells derived from the nervous system; evidence for co-regulation and functional homology with c-myc and n-myc. [Manuscript]</p><p>IV. Wennborg A, Classon M, Klein G, von Gabain A (1995). Downregulation of c-myc expression after heat shock in human B-cell lines is independent of 5 mRNA sequences. Biol Chem Hoppe Seyler. 376(11): 671-80. <br><a href="https://pubmed.ncbi.nlm.nih.gov/8962676">https://pubmed.ncbi.nlm.nih.gov/8962676</a><br><br></p><p>V. A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control. (1995). Wennborg A, Sohlberg B, Angerer D, Klein G, von Gabain A. Proc Natl Acad Sci U S A. 92(16): 7322-6. <br><a href="https://pubmed.ncbi.nlm.nih.gov/7638189">https://pubmed.ncbi.nlm.nih.gov/7638189</a><br><br></p>