Abstract
Posttranscriptional mechanisms play important roles in insulin receptor gene regulation; variability in cellular insulin receptor number and the growth arrest-mediated increases in insulin receptor mRNA are secondary to changes in insulin receptor mRNA stability. Therefore, further characterization of the pathways and kinetics of insulin receptor mRNA degradation were investigated. The insulin receptor mRNA in the insulin receptor-rich Hep G2 cells is more stable compared with the insulin receptor-sparse MCF-7 cells. Growth arrest results in a significant rise in insulin receptor mRNA in both cell lines. The increase in mRNA is caused by changes in mRNA stability. The half-life of the insulin receptor mRNA in growth-arrested cells is approximately three times that of proliferating cells. The insulin receptor gene contains four polyadenylation sites that produce four species of mRNA of 5.4, 6.9, 8.0, and 9.4 kilobases (kb). The mRNA species are not coordinately regulated. The ratio of the most abundant species (9.4/6.9) is significantly larger in growth-arrested cells compared with proliferating cells. By utilizing a specific cDNA probe for the 9.4-kb mRNA species, it was determined that the diminished 9.4/6.9 ratio in proliferating cells was caused by a more rapid rate of the 9.4-kb mRNA degradation. The kinetics of insulin receptor mRNA degradation were investigated. Insulin receptor mRNA levels were reduced to 56% of their base line within 6 h when growth-arrested cells were stimulated to proliferate; protein inhibition with cycloheximide completely inhibited the decline in insulin receptor mRNA.
Highlights
Posttranscriptional mechanisms play important roles in insulin receptor gene regulation; variabilityin cellular insulin receptor number and the growth arrest-mediated increases in insulin receptomr RNA are secondary to changes in insulin recepmtoRr NA stability
In the present study we have investigated the regulation of the insulin receptor gene in various stages of cell proliferation with particular emphasis on the role of mRNA stability
Because posttranscriptional mechanisms may determine the variability of cellular insulin receptor number, we have compared the regulation of the insulin receptor gene in a cell line with abundant receptors (Hep G2) and a cell line with few receptors (MCF-7).We havefound that growth arrest in both cell lines resultsin an increase in insulin receptor mRNA because of a specific increase in insulin receptor mRNA stability
Summary
The kineticsof insulin receptor mRNA degradationwereinvestigated.Insulinreceptor mRNA levels were reduced to 56%of their base line within h when growth-arrested cells were stimulated to proliferate; protein inhibition with cycloheximide completely inhibited the decline in insulin receptor mRNA. In the present study we have investigated the regulation of the insulin receptor gene in various stages of cell proliferation with particular emphasis on the role of mRNA stability. Because posttranscriptional mechanisms may determine the variability of cellular insulin receptor number, we have compared the regulation of the insulin receptor gene in a cell line with abundant receptors (Hep G2) and a cell line with few receptors (MCF-7).We havefound that growth arrest in both cell lines resultsin an increase in insulin receptor mRNA because of a specific increase in insulin receptor mRNA stability. We predicted that digestion of the 3”flanking region with the restriction enzyme BglII would result in a 1.3-kb fragment that would hybridize to the 9.4-kb insulin receptor mRNA but not to the smaller insulin receptor mRNA species
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