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Endoribonuclease RNase £ 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.

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.

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.Fibronectins (FNs) are large glycoproteins implicated in a wide variety of cellular properties including cell adhesion, morphology, migration, differentiation, and transformation (21). FNs exist in extracellular matrices (cellular FN) and in soluble form in body fluids (plasma FN) and are composed of two similar but nonidentical subunits held together by disulfide bonds. Alternative splicing of the primary transcript of a single gene located on human chromosome 2 generates numerous distinct FN transcripts which account for the individual subunits and for the plasma and cellular isoforms. Loss of cell surface FN is a hallmark of many oncogenically transformed cells and has been correlated with the acquisition of tumorigenic and metastatic potentials (21). This effect has been observed in cells transformed with a variety of oncogenes; however, there is still little known about the mechanisms by which oncogenes affect FN biosynthesis. In two cases, transformation of chick embryo fibroblasts by src and growth stimulation of rat embryo fibroblasts by ElA, the primary defects in FN production are reported to be at the level of transcription (31, 45).The ras family of genes encode related 21-kDa plasma membrane-associated proteins that bind guanine nucleotides and are involved in signal transduction and cell growth (2). ras oncogenes are mutated in many types of human tumors. As a result of mutation, there is a loss of control of...

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