Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes

Abstract

Background Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown. Methods Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4′,6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation. Results We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation. Conclusions We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues. Graphical Abstract

Two distinctTrypanosomaeIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

“…This mechanism is unlikely to exist in trypanosomatids because all mRNAs have a SL and ribosomal-protein encoding mRNAs have very short 5’ UTRs lacking TOP motifs [45], [83]. Recently, ZC3H41 and its associated protein (Z41AP) have been shown to co-precipitate ribosomal-protein encoding mRNAs in a manner sensitive to nutritional stress [89]. We do see association of ZC3H41 and Z41AP with eIF4E4 (native and tagged).…”