Evidence for translational control of β-tubulin synthesis during differentiation ofLeishmania donovani

Abstract

Tubulin biosynthesis was rapidly induced during transformation of the mammalian (amastigote) stage of the kinetoplastid parasiteLeishmania donovanito flagellated promastigotes. However, transcription of β-tubulin genes occurred constitutively, as judged by nascent RNA synthesis in isolated nuclei and Northern blotting of steady-state mRNA. Two mRNA species of 2.2 and 2.4 kb were shared by the two cell-types, while a third 2.6 kb species, constituting about 20% of the total, was present in large amounts in promastigotes. RNase protection experiments demonstrated sequence micro-heterogeneity in the 5′-untranslated region, the pattern of which was identical in promastigotes and amastigotes. By primer extension assays, heterogeneity in the 5′-terminal cap structure of amastigote β-tubulin mRNA and differential pausing of reverse transcriptase within the mini-exon leader region were detected. These differences correlated with enhanced translational efficiency of tubulin mRNA from promastigotes in a rabbit reticulocyte lysate system. The results indicate that translational control plays a major role in tubulin induction duringL. donovanidifferentiation.