Elements in the 3′ Untranslated Region of Procyclin mRNA Regulate Expression in Insect Forms of Trypanosoma brucei by Modulating RNA Stability and Translation

Abstract

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3′ untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3′ UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3′ UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an approximately 2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t_1/2) of the mRNA was reduced from approximately 50 min (the t_1/2 of the wild-type 3′ UTR) to <15 min, whereas removal of the LII element resulted in an increased t_1/2 of approximately 100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.