Pseudouridines on Trypanosoma brucei mRNAs are developmentally regulated: Implications to mRNA stability and protein binding

• Published in: Molecular microbiology 2021-09, Vol.116 (3), p.808-826
• Main Authors: Rajan, K. Shanmugha, Adler, Katerina, Madmoni, Hava, Peleg‐Chen, Dana, Cohen‐Chalamish, Smadar, Doniger, Tirza, Galili, Beathrice, Gerber, Doron, Unger, Ron, Tschudi, Christian, Michaeli, Shulamit
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; African trypanosomiasis; Animals; Gene Expression Regulation; High-Throughput Screening Assays - methods; Homology; Insects; Intramolecular Transferases - genetics; Intramolecular Transferases - metabolism; mRNA stability; Parasites; Protein Binding; Proteins; Protozoa; pseudouridine (Ψ); Pseudouridine - genetics; Pseudouridine - metabolism; Pseudouridylation; PUS enzymes; RNA modification; RNA Stability; RNA, Messenger - metabolism; RNA-binding protein; RNA-Binding Proteins - metabolism; Stability; Transcription; tRNA; Trypanosoma brucei; Trypanosoma brucei brucei - genetics; Trypanosoma brucei brucei - metabolism; trypanosomes; Vector-borne diseases; Ψ protein interaction; Ψ‐seq on mRNAs
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.14774

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high‐throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3′ UTR of valyl‐tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Ψ affects the stability of this mRNA, proteins that bind to the 3′ UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3′ UTR was stronger when lacking Ψ compared to transcripts carrying the modification, suggesting that Ψ can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Ψ modification on mRNA adds an additional level of regulation to the dominant post‐transcriptional control in these parasites. Trypanosomes possess hundreds of pseudouridines (Ψs) on mRNAs, some of which are guided by pseudouridine synthase (PUS) enzymes. Ψs are developmentally regulated on a subset of mRNAs. The Ψ studied in this study is located in the binding site of an RNA binding protein (RBSR1) which its binding is dependent on the RNA modification and controls mRNA stability. Thus, mRNA pseudouridylation could be considered as an additional mechanism controlling mRNA fate.