The universal N⁶-threonylcarbamoyladenosine (t⁶A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t⁶A stabilizes structural configurations of the anticodon stem loop, promotes anticodon–codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t⁶A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO₃⁻/CO₂ and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD–TsaB –TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t⁶A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway–Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t⁶A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t⁶A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure–function relationship from perspectives of conservation and diversity.

We present an in‐depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three‐dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Cholangiocarcinoma (CCA) is a bile duct malignancy with a dismal prognosis. This study systematically investigated the role of the ribosomal protein S6 (RPS6) gene, which is dependent in CCA. We found that RPS6 upregulation in CCA tissues was correlated with a poor prognosis. Functional investigations have shown that alterations in RPS6 expression, both gain- and loss-of function could affect the proliferation of CCA cells. In xenograft tumor models, RPS6 overexpression enhances tumorigenicity, whereas RPS6 silencing reduces it. Integration analysis using RNA-seq and proteomics elucidated downstream signaling pathways of RPS6 depletion by affecting the cell cycle, especially DNA replication. Immunoprecipitation followed by mass spectrometry has identified numerous spliceosome complex proteins associated with RPS6. Transcriptomic profiling revealed that RPS6 affects numerous alternative splicing (AS) events, and combined with RNA immunoprecipitation sequencing, revealed that minichromosome maintenance complex component 7 (MCM7) binds to RPS6, which regulates its AS and increases oncogenic activity in CCA. Targeting RPS6 with vivo phosphorodiamidate morpholino oligomer (V-PMO) significantly inhibited the growth of CCA cells, patient-derived organoids, and subcutaneous xenograft tumor. Taken together, the data demonstrate that RPS6 is an oncogenic regulator in CCA and that RPS6-V-PMO could be repositioned as a promising strategy for treating CCA.

The structure and function of transfer RNAs (tRNAs) are highly dependent on post-transcriptional chemical modifications that attach distinct chemical groups to various nucleobase atoms at selected tRNA positions via enzymatic reactions. In all three domains of life, the greatest diversity of chemical modifications is concentrated at positions 34 and 37 of the tRNA anticodon loops. N⁶-threonylcarbamoyladenosine (t⁶A) is an essential and universal modification occurring at position 37 of tRNAs that decode codons beginning with an adenine. In a subset of tRNAs from specific organisms, t⁶A is converted into a variety of hypermodified forms, including cyclic N⁶-threonylcarbamoyladenosine (ct⁶A), hydroxy-N⁶-threonylcarbamoyladenosine (ht⁶A), N⁶-methyl-N⁶-threonylcarbamoyladenosine (m⁶t⁶A), 2-methylthio-N⁶-threonylcarbamoyladenosine (ms²t⁶A) and 2-methylthio-cyclic N⁶-threonylcarbamoyladenosine (ms²ct⁶A). The tRNAs carrying t⁶A or one of its hypermodified derivatives are dubbed as the t⁶A family. The t⁶A family modifications pre-organize the anticodon loop in a conformation that enhances binding to the cognate mRNA codons, thereby promoting translational fidelity. The dysfunctional installation of modifications in the tRNA t⁶A family leads to translation errors, compromises proteostasis and cell viability, interferes with the growth and development of higher eukaryotes and is implicated in several human diseases, such as neurological disorders, mitochondrial encephalomyopathies, type 2 diabetes and cancers. In addition, loss-of-function mutations in KEOPS complex-the tRNA t⁶A-modifying enzyme-are associated with shortened telomeres, defects in DNA damage response and transcriptional dysregulation in eukaryotes. The chemical structures, the molecular functions, the known cellular roles and the biosynthetic pathways of the t⁶A tRNA family are described by integrating and linking biochemical and structural data on these modifications to their biological functions.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits―Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N⁶-threonylcarbamoyladenosine (t⁶A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t⁶A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t⁶A-catalytic KEOPS–tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t⁶A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t⁶A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

N⁶-threonylcarbamoyladenosine (t⁶A) is a post-transcriptional modification found uniquely at position 37 of tRNAs that decipher ANN-codons in the three domains of life. tRNA t⁶A plays a pivotal role in promoting translational fidelity and maintaining protein homeostasis. The biosynthesis of tRNA t⁶A requires members from two evolutionarily conserved protein families TsaC/Sua5 and TsaD/Kae1/Qri7, and a varying number of auxiliary proteins. Furthermore, tRNA t⁶A is modified into a cyclic hydantoin form of t⁶A (ct⁶A) by TcdA in bacteria. In this work, we have identified a TsaD–TsaC–SUA5–TcdA modular protein (TsaN) from Pandoraviruses and determined a 3.2 Å resolution cryo-EM structure of P. salinus TsaN. The four domains of TsaN share strong structural similarities with TsaD/Kae1/Qri7 proteins, TsaC/Sua5 proteins, and Escherichia coli TcdA. TsaN catalyzes the formation of threonylcarbamoyladenylate (TC-AMP) using L-threonine, HCO₃⁻ and ATP, but does not participate further in tRNA t⁶A biosynthesis. We report for the first time that TsaN catalyzes a tRNA-independent threonylcarbamoyl modification of adenosine phosphates, leading to t⁶ADP and t⁶ATP. Moreover, TsaN is also active in catalyzing tRNA-independent conversion of t⁶A nucleoside to ct⁶A. Our results imply that TsaN from Pandoraviruses might be a prototype of the tRNA t⁶A- and ct⁶A-modifying enzymes in some cellular organisms.