A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Kathrin Bajak,Kevin Leiss,Christine Clayton,Esteban Erben

doi:10.1074/jbc.ra120.014346

Abstract

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

Highlights

Trypanosoma brucei is a unicellular eukaryote that proliferates in the blood and tissue fluids of mammals and in the digestive system of tsetse flies
Several of the identified RNA-binding protein (RBP) are essential for stagespecific gene expression and/or have developmentally regulated expression or modification, only a small fraction of them exhibited a clear effect on expression when tethered to the mRNA reporter
The regulation of gene expression in trypanosomatids occurs mainly at the posttranscriptional level; RNA-binding proteins are fundamental for the regulation of mRNA and protein abundance

Summary

Introduction

Trypanosoma brucei is a unicellular eukaryote that proliferates in the blood and tissue fluids of mammals and in the digestive system of tsetse flies. Tb927.8.1500 has four phosphorylated serines [12, 13] and three phosphothreonines (Fig. S3), one of Tethering of ZC3H5 complex components inhibits reporter gene expression Consistent with this, results of a preliminary experiment suggested that all four proteins migrate predominantly in the free fractions of a sucrose gradient, rather than with monosomes or polysomes (Fig. S7).

Results

Conclusion