Citation: Fatica, T.; Naas, T.; Liwak, U.; Slaa, H.; Souaid, M.; Frangione, B.; Kattini, R.; Gaudreau-Lapierre, A.; Trinkle-Mulcahy, L.; Chakraborty, P.; et al. TRNT-1 Deficiency Is Associated with Loss of tRNA Integrity and Imbalance of Distinct Proteins. Genes 2023, 14, 1043. https://doi.org/10.3390/ genes14051043 Academic Editor: Jia-Yu Chen Received: 27 March 2023 Revised: 1 May 2023 Accepted: 4 May 2023 Published: 5 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article TRNT-1 Deficiency Is Associated with Loss of tRNA Integrity and Imbalance of Distinct Proteins Thet Fatica 1,† , Turaya Naas 2,† , Urszula Liwak 2 , Hannah Slaa 2 , Maryam Souaid 1 , Brianna Frangione 1 , Ribal Kattini 1 , Antoine Gaudreau-Lapierre 3 , Laura Trinkle-Mulcahy 3 , Pranesh Chakraborty 2 and Martin Holcik 1, * 1 Department of Health Sciences, Carleton University, Ottawa, ON K1S 5B6, Canada 2 Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada 3 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada * Correspondence: martinholcik@cunet.carleton.ca † These authors contributed equally to this work. Abstract: Mitochondrial diseases are a group of heterogeneous disorders caused by dysfunctional mitochondria. Interestingly, a large proportion of mitochondrial diseases are caused by defects in genes associated with tRNA metabolism. We recently discovered that partial loss-of-function mutations in tRNA Nucleotidyl Transferase 1 (TRNT1), the nuclear gene encoding the CCA-adding enzyme essential for modifying both nuclear and mitochondrial tRNAs, causes a multisystemic and clinically heterogenous disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay; SIFD). However, it is not clear how mutations in a general and essential protein like TRNT1 cause disease with such clinically broad but unique symptoma- tology and tissue involvement. Using biochemical, cell, and mass spectrometry approaches, we demonstrate that TRNT1 deficiency is associated with sensitivity to oxidative stress, which is due to exacerbated, angiogenin-dependent cleavage of tRNAs. Furthermore, reduced levels of TRNT1 lead to phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha (eIF2α), increased reactive oxygen species (ROS) production, and changes in the abundance of distinct proteins. Our data suggest that the observed variable SIFD phenotypes are likely due to dysregulation of tRNA maturation and abundance, which in turn negatively affects the translation of distinct proteins. Keywords: SIFD; TRNT1; tiRNA; CCA adding; oxidative stress; SILAC 1. Introduction The maturation of tRNAs (both mitochondrial and cytoplasmic) is a multi-step pro- cess that requires 5 ′ and 3 ′ processing, extensive base modifications, CCA addition, and aminoacylation. For cytoplasmic tRNAs, these steps occur in a precise order and are per- formed in distinct cellular compartments; in contrast, all mt tRNA processing steps occur in mitochondria [1]. The CCA addition is performed by a unique CCA-adding enzyme, tRNA nucleotidyltransferase (TRNT1 in humans or CCA1 in yeast). TRNT1 encodes the only human CCA-adding enzyme, an RNA polymerase required for the post-transcriptional, template-independent addition of two cytosines and one adenosine to the 3 ′ end of both cytosolic and mitochondrial tRNAs [2]. This 3 ′ addition is required for aminoacylation, correct positioning on the ribosome, and subsequent protein synthesis [3]. Following the addition, the CCA trinucleotide acts as an anti-determinant for 3 ′ endoribonuclease activity [4]. The 3 ′ CCA tRNA terminus is subject to frequent cleavage or degradation, and TRNT1 also functions to maintain and repair previously added CCA sequences [5]. Importantly, TRNT1 can discriminate against tRNA backbone damage, keeping damaged tRNAs from incorporation into the translation machinery [6]. We have previously reported that partial loss-of-function mutations in TRNT1 cause a novel disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic Genes 2023, 14, 1043. https://doi.org/10.3390/genes14051043 https://www.mdpi.com/journal/genes