Uteroglobin Messenger RNA: Translation in vitro1

Abstract

The messenger RNA (mRNA) coding for the progesterone-induced protein uteroglobin has been extracted from endometrial tissue of rabbits in early pregnancy and enriched by binding to oligo-dT-cellulose. After translation in a cell-free system derived from wheat germ, total mRNA activity was assessed by measuring the incorporation of ³⁵S-methionine into TCA-precipitable peptides and specific mRNA activity by immunoprecipitation with specific uteroglobin antibodies purified by affinity chromatography. Approximately 85 percent of total mRNA activity was recovered after dT-cellulose chromatography, 10 percent in the bound fraction and 75 percent in the unbound RNA, suggesting that the majority of endometrial mRNA species lacks poly A sequences of longer than about 20 residues. No poly A could be detected by ³H-poly U hybridization in the unbound fraction. In contrast, 69 percent of total mRNA activity was present in dT-bound RNA from rabbit liver. The immunoprecipitable cell-free translation products of endometrial dT-RNA gave a single peak of radioactivity on electrophoresis in 15 percent polyacrylamide gels containing sodium dodecyl sulfate. The peak was completely displaced by the addition of an excess of authentic nonradioactive uteroglobin to the immunoprecipitation reaction and was absent from products of translation without added endometrial RNA. The cell-free product migrated more slowly than authentic uteroglobin, suggesting the synthesis of a precursor protein. No uteroglobin mRNA could be detected in dT-bound RNA from rabbit liver. The proportion of uteroglobin mRNA in endometrial dT-bound RNA reached a peak on Day 4 of pregnancy and declined subsequently to nonpregnant levels on Day 8, a pattern similar to that of uteroglobin secretion.