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

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. This makes the parasites exquisitely reliant on post-transcriptional 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 mRNAs control degradation and translation, through interactions with RNA-binding proteins. However, the mechanisms by which the RNA-binding proteins act are mostly unknown. We conducted a genome wide "tethering" screen and identified nearly 300 proteins that increase or decrease gene expression when attached to a reporter mRNA. Those that decreased gene expression were enriched for proteins with RNA-binding domains. In the current project, we aim to determine the functions of some of these proteins. We will start with several candidates and focus on a sub-set depending on initial results. We will choose proteins that gave strong down-regulation in the tethering screen and are essential for growth in at least one of the easily cultivated trypanosome life-cycle stages. The subcellular location of the protein will be determined, and cytosolic proteins will be selected. Using polysome profiling and Northern blot, we will find out whether the effect of the artificially attached (tethered) protein is primarily on translation or stability of the reporter mRNA. For selected proteins, the effects of depleting the protein (by RNAi) on the transcriptome and translation profiles will be determined: specifically affected mRNAs are potential targets. The RNA sequences bound by the protein will be determined by Motif searches on mRNAs affected by the RNAi, and reporter assays using mRNAs with and without candidate motifs. After inhibition of mRNA decay, we will attempt to find in vivo bound RNAs using UV-cross linking, affinity purification, then RNA sequencing. Production of soluble protein and in vitro RNA-binding assays are an alternative. To find the mechanism of action, protein-protein interactions will be investigated by yeast-2-hybrid assays and pull-downs. The roles of interactions in RNA regulation will be determined by structure-function analyses.This project is part of a longer-term plan which includes the characterization of proteins that increase expression, and of proteins that were positive in the tethering screen but do not have any domains that link them to RNA metabolism. The ultimate aim is to obtain a quantitative understanding of the network of post-transcriptional regulation in trypanosomes. We hope also to discover novel regulatory mechanisms, some which may also be present in other organisms.

• DFG Programme: Research Grants