Since protein binding to the 3' end of
mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in
glucagon-induced
phosphoenolpyruvate-carboxykinase-
mRNA (PCK) levels, in the absence and presence of
insulin, was correlated with the time course of changes in the binding of cytosolic
protein from 24-h cultured rat hepatocytes to the 3' end of PCK
mRNA. PCK-
mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility-shift assay. In 24-h cultured rat hepatocytes, binding of cytosolic
protein to the PCK-
mRNA 3' end and PCK-
mRNA levels were increased to a transient maximum at 2 h and 2-4 h, respectively, by a 1-nM
glucagon treatment, added with a change of medium. 100 nM
insulin, added simultaneously with
glucagon, reduced the
glucagon-induced maximum of protein binding by 80% and the increase of PCK
mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by
insulin. 100 nM
insulin, added 1 h after
glucagon, reversed protein binding to the 3' end of PCK
mRNA to nearly initial levels within 1 h and impaired the
glucagon-induced increase in PCK-
mRNA levels by 30%. The transcriptional inhibitor
cordycepin, added 1 h after
glucagon, did not prevent the further increase in
glucagon-enhanced protein binding nor its reversal by
insulin. It did, however, prevent a further significant increase in PCK
mRNA. Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK-
mRNA 3' end. The proximal 466 bases of the PCK-
mRNA 3' end as well as the 1050 bases of the histone-H1(0)-mRNA 3' end and the 1200 bases of the
arylsulfatase-A-
mRNA 3' end also bound cytosolic
protein(s), but this protein binding was not altered by treatment with
glucagon or
insulin. The 3' end of PCK,
arylsulfatase A and
H1(0)
mRNA exhibited strong binding of cytosolic
protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat
hepatoma cells. Cytosolic
protein(s) from spleen showed weak binding and
proteins from HeLa and U937
tumor cells did not bind. Protein binding was most prominent with the 3' end of PCK
mRNA and cytosolic extracts from liver.(ABSTRACT TRUNCATED AT 400 WORDS)