UTILIZATION OF MULTIPLE POLYADENYLATION SIGNALS IN THE HUMAN RHOA PROTOONCOGENE

Little is known regarding the regulation of expression of the RHOA pro tooncogene, a member of the family of genes encoding Ras-related GTP-b inding proteins. We have previously reported that the 3' untranslated region (UTR) of RHOA was contained within a genomic sequence which fla nked the 5' end of the human glutathione peroxidase 1-encoding gene [J .A. Moscow et al., J. Biol. Chem. 267 (1992) 5949-5958]. Our previous studies revealed the presence of multiple (1.8 and 1.5 kb) RHOA mRNA s pecies in breast cancer cell lines and of three putative polyadenylati on signals in the RHOA 3' UTR. In this report, we have isolated severa l RHOA cDNAs from a multidrug-resistant MCF-7 human breast cancer cell line. Sequence analyses of these RHOA cDNA clones indicate that multi ple polyadenylation signals are used to terminate RHOA transcripts. RN ase-protection analysis demonstrated that all three polyadenylation si gnals are utilized in breast cancer cell lines and RNA stability studi es demonstrated that RHOA RNA species with different 3' ends have equi valent stability. Since little is known about the RNA expression of RH OA in human tumors, and since both activated and non-activated RHOA ge ne possess transformation potential, we analyzed RHOA mRNA in lung and colon tumors by Northern blot and RNase-protection analyses. In all e ight lung tumors examined, RHOA RNA levels were decreased relative to the level in normal surrounding tissue, whereas RHOA expression was de creased in only two of six colon tumors. We also found that lovastatin -induced cell cycle arrest resulted in increased RHOA RNA expression i n breast cancer cell lines. RNase-protection analysis of RHOA RNA from these tumor and cell line specimens demonstrated that the relative ab undance of RNA transcripts utilizing these three polyadenylation signa ls did not vary with total RHOA mRNA levels.