A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

A nuclear post-transcriptional mechanism mediates the induction of fibronectin by glucocorticoids.

Treatment of the human fibrosarcoma cell line HT-1080 with glucocorticoids results in the induction of fibronectin (FN) protein and mRNA synthesis. We tested the contribution of transcriptional and post-transcriptional mechanisms in the regulation of FN by the synthetic glucocorticoid dexamethasone (DEX). Using nuclear run-on experiments, we found that the DEX-dependent induction of FN occurs primarily at the post-transcriptional level. The half-life of total FN mRNA was not affected by hormone treatment indicating that the induction of FN gene expression is not due to stabilization of the mature message. Interestingly, the induction by DEX was present at the level of nuclear FN RNA. We found that polyadenylation and alternative splicing of the ED-B domain of the FN transcript were not affected by glucocorticoid treatment. However, DEX was found to increase the steady-state level of unspliced FN transcript. Our data indicate that DEX exerts its effect on FN expression predominantly at the post-transcriptional level by a mechanism that, unlike most examples of post-transcriptional regulation by glucocorticoids, acts in the nucleus. Furthermore, they suggest that glucocorticoids activate a mechanism to stabilize the unspliced FN RNA. In an attempt to localize the FN RNA sequences mediating the DEX-dependent induction, we performed transfection analyses of FN minigene constructs. We suggest that the DEX-dependent regulatory elements are located in the introns since no such elements were found in the 8 kb FN mRNA.

Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site.

Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.

mRNA degradation in procaryotes.

The fast turnover of mRNA permits rapid changes in the pattern of gene expression. In procaryotes, many enzymes involved in mRNA degradation have been identified and some of these endo- and exo-ribonucleases are now being intensively studied. Some of the structural features of mRNA that influence decay rates have also recently been defined. Although important components of the decay pathway are still elusive, a coherent and simple model for mRNA decay has emerged in the last few years.