Quantitative and qualitative changes in mRNA translation occur in tumor cells and support cancer progression and metastasis. Posttranscriptional modifications of transfer RNAs (tRNAs) at the wobble uridine 34 (U34) base are highly conserved and contribute to translation fidelity. Here, we show that ELP3 and CTU1/2, partner enzymes in U34 mcm5s2-tRNA modification, are up-regulated in human breast cancers and sustain metastasis. Elp3 genetic ablation strongly impaired invasion and metastasis formation in the PyMT model of invasive breast cancer. Mechanistically, ELP3 and CTU1/2 support cellular invasion through the translation of the oncoprotein DEK. As a result, DEK promotes the IRES-dependent translation of the proinvasive transcription factor LEF1. Consistently, a DEK mutant, whose codon composition is independent of U34 mcm5s2-tRNA modification, escapes the ELP3- and CTU1-dependent regulation and restores the IRES-dependent LEF1 expression. Our results demonstrate that the key role of U34 tRNA modification is to support specific translation during breast cancer progression and highlight a functional link between tRNA modification– and IRES-dependent translation during tumor cell invasion and metastasis.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin‐related modifier 1 (Urm1) pathway is responsible for the synthesis of 2‐thiolated wobble uridine (U ₃₄ ). During the key step of the modification cascade, the E1‐like activating enzyme ubiquitin‐like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C‐terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1‐COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin‐like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin‐like proteins (UBL) and sulfur‐relay systems. Here, we report the crystal structures of full‐length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C‐terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self‐conjugation with its own product, namely activated Urm1‐COSH.

Eukaryotic ubiquitin-like proteins (UBLs) have evolved from prokaryotic sulfur-carrier proteins (SCPs). Ubiquitin related modifier 1 (Urm1) shares biochemical and structural features of UBLs and SCPs and is essential for 2-thiolation of cytoplasmic tRNA. This chemical modification of wobble uridine is highly conserved amongst species and is achieved via Urm1 thiocarboxylation by the non-canonical ubiquitin activating 4 enzyme (Uba4), which contains an E1- and a Rhodanese (RHD) domain. While the RHD catalyzes the last step in Urm1-thiocarboxylate formation, the previous steps in Urm1 activation and the interplay between the two domains have remained elusive. To define the underlying mechanism, we established an Urm1 in vitro thiocarboxylation assay, which combined with structure-function and chemical profiling analyses revealed a critical thioester linkage between Urm1 and Uba4 residue Cys225. This linkage is indispensable for the Urm1 intramolecular transfer between the two domains of Uba4 and it is thus, essential for tRNA thiolation in vivo. These findings contribute to a deeper understanding of UBL evolution.