Post-transcriptional repression of gene expression in trypanosomes: roles of RNA-binding proteins in translation and mRNA decay

• Applicant: Professorin Dr. Christine Elizabeth Clayton
• Subject Area: General Genetics and Functional Genome Biology ; Parasitology and Biology of Tropical Infectious Disease Pathogens ; Cell Biology
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 268445533

• Final Report Year: 2019

Final Report Abstract

Post-transcriptional mechanisms are vital for regulation of gene expression in all organisms. Trypanosomes are excellent models for the study of mRNA degradation because there is no control of RNA polymerase II activity at the level of individual open reading frames. Nevertheless, levels of different mRNAs and proteins can differ by several orders of magnitude, and also change during transitions between different life-cycle stages. To achieve this, the parasites rely on posttranscriptional mechanisms, with extensive regulation of mRNA decay rates and translation. We have identified most of the enzymes and complexes that are required for trypanosome mRNA degradation, and measured mRNA decay rates transcriptome-wide. Results so far suggest that usually, the 3'-untranslated regions of trypanosome mRNAs control degradation and translation, through interactions with RNA-binding proteins. To identify proteins that could act as posttranscriptional regulators, we conducted a genome wide "tethering" screen. A library that expressed trypanosome protein fragments fused to a bacteriophage RNA-binding peptide was expressed in trypanosomes that expressed a reporter RNA containing the peptide-binding motif in the 3'-untranslated region. We identified nearly 300 proteins that increased or decreased gene expression when attached to the reporter mRNA. Those that decreased gene expression were highly enriched for proteins with RNA-binding domains. The activities of the full-length proteins were confirmed in a targeted screen. In the current project, we aim to determine the functions of some of these proteins, which were selected based on their RNA binding and ability to suppress expression in the tethering screen. We also focused on proteins that appeared to be essential in mammalian-infective trypanosomes, based on the results of a published RNAi screen.

Publications

• (2017) Conversion of procyclic-form Trypanosoma brucei to the bloodstream form by transient expression of RBP10. Mol Biochem Parasitol 216C, 49-51
  Mugo E, Egler F, Clayton C
  
• (2017) Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei. PLoS Pathogens 13, e1006560
  Mugo E and Clayton C
  
• (2017) The role of RBP10, a key post-transcriptional regulator in the development of Trypanosoma brucei
  Elisha Mugo
  
• (2017). The role of the zinc finger protein ZC3H32 in bloodstreamform Trypanosoma brucei. PLoS ONE 12, e0177901
  Klein C, Terrao M, Clayton C
  
• (2018) The suppressive mRNA cap-binding-complex factor 4EIP is required for normal differentiation of Trypanosoma brucei. Nucleic Acids Res 46, 8993-9010
  Terrao, M, Marucha, KM, Mugo, E, Braun, J, Droll, D, Minia, I, Egler, F, Clayton, C