Our laboratory and others have reported that treatment of GT1-7 cells with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits transcription of the pro-GnRH gene and decreases messenger RNA (mRNA) levels. We were interested in whether translation of the existing GnRH mRNA decreases in parallel with these other indexes of biosynthesis after PMA treatment. GT1-7 cells were treated with PMA (100 nM) or vehicle for 0, 1, or 4 h. The cytosolic ribosome-associated RNA was isolated and layered on a continuous (10-40%) sucrose gradient, and fractions were analyzed for the distribution of ribosome-associated GnRH mRNA through the gradient by ribonuclease protection assay. The mRNA found in the lighter fractions is associated with fewer ribosomes per RNA, suggesting that these fractions are translated less efficiently, and RNA recovered from heavier fractions has a higher number of ribosomes per mRNA, representing mRNA that is more actively translated. We found that the distribution of the ribosome-associated GnRH mRNA was shifted into lighter fractions (i.e. fewer ribosomes per mRNA) after PMA treatment, indicating that a decrease in the translational efficiency of GnRH mRNA occurs after PMA treatment. Thus, PMA exerts inhibitory effects on translation of GnRH mRNA as well as on gene transcription, mRNA stability, and mRNA levels.
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