Poster abstracts
Poster number 49 submitted by Lankani Gunaratne
A Surprising Role of Trypanosoma brucei La (TbLa) in controlling Queuosine levels in tRNA
G. Lankani Gunaratne (Ohio State Biochemistry Program, The Ohio State University), Jeremy C. Henderson (Department of Microbiology, The Ohio State University), Ananth Casius (Department of Microbiology, The Ohio State University), Eva Hegedsov, Sneha Kulkarni, Zdenk Paris (Institute of Parasitology, Biology Centre, Czech Academy of Sciences, esk Budjovice Budweis, Czech Republic), Alan C. Kessler, Richard J. Maraia (Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health), Juan D. Alfonzo (Department of Microbiology, The Ohio State University)
Abstract:
La is a ubiquitous RNA-binding protein found in almost all eukaryotes. Although in many eukaryotes La is essential, its role in eukaryotic cells is not completely clear. Trypanosoma brucei, the causative agent of African Trypanosomiasis in humans, encodes a ‘classical’ genuine La protein (TbLa) with a La motif closely followed by an RNA recognition motif (RRM1), an additional RRM (RRM2α) and a C terminal short basic motif (SBM) along with a nuclear localization signal (NLS), and although La is primarily nuclear, in many organisms, including T. brucei, a small portion of La localizes to the cytoplasm. In T. brucei, like in most eukaryotes, position 34 of tRNATyr, -Asp, -Asn, and -His is modified with queuosine (Q), a 7-deaza-derivative of guanosine. The steady-state levels of queuosine-modified tRNA (Q-tRNA) are approximately 40-60% in procyclic form (insect form) of T. brucei, but these levels can dynamically fluctuate depending on growth conditions. However, the mechanism(s) controlling Q levels are currently not well understood. Previously, we showed that increased tRNA nuclear retention leads to higher levels of Q-tRNA. This is because tRNA guanine transglycosylase (TGT), the enzyme responsible for Q incorporation into tRNA, is a nuclear enzyme in T. brucei and presumably, there is competition between nuclear tRNA export and Q formation. In this work, we demonstrate that Q-tRNA levels are also influenced by La, but surprisingly changes in Q levels are not due to the increased dwell time of tRNA in the nucleus following La RNAi. We have also constructed a series of myc-tagged recoded La mutants with each of the RRMs, SBM, and/or the NLS deleted. Through in vivo expression of individual domain-truncated La mutants in endogenous La RNAi cells, we show that while the deletion of RRM1 and NLS causes an increase of Q-tRNA, deletion of RRM2α or SBM does not cause a significant change in Q-tRNA levels. Investigating how the Q-tRNA dynamics affect translation and biological processes in T. brucei will reveal potential new functions of TbLa.
References:
Blewett,N.; Maraia,R. La Involvement in tRNA and Other RNA Processing Events Including Differences among Yeast and Other Eukaryotes. Biochim. Biophys. Acta Gene Regul. Mech. 2018
Maraia,R.; Mattijssen,S.; Cruz-Gallardo,I.; Conte,M. The La and Related RNA-Binding Proteins (LARPs): Structures, Functions, and Evolving Perspectives. Wiley Interdiscip. Rev. RNA 2017
Kulkarni,S.; Rubio,M.A.; Hegedusová,E.; Ross,R.; Limbach,P.; Alfonzo,J.; Paris,Z. Preferential Import of Queuosine-Modified tRNAs into Trypanosoma brucei Mitochondrion Is Critical for Organellar Protein Synthesis. Nucleic Acids Res. 2021
Dixit,S.; Kessler,A.; Henderson,J.; Pan,X.; Zhao,R.; D’Almeida,G.S.; Kulkarni,S.; Rubio,M.A.; Hegedűsová,E.; Ross,R.; Limbach,P.; Green,B.; Paris,Z.; Alfonzo,J.D. Dynamic Queuosine Changes in tRNA Couple Nutrient Levels to Codon Choice in Trypanosoma brucei. Nucleic Acids Res. 2021
Keywords: La protein, tRNA modifications, Trypanosoma brucei