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Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate–rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3′ untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However, it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here, we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors, ATG8-like proteins, or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy, while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening, protein engineering, and structural analysis, this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation.

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer. Lineage-restricted transcription factor PAX8 is oncogenic in ovarian cancer cells. Here the authors show that PAX8 interacts and recruits a splice variant of the MECOM locus PRDM3 to control the gene expression module involved in adhesion and extracellular matrix, and consequently promotes ovarian tumorigenesis.

An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein–ligand interactions are characterized by 2D [ 15 N, 1 H]- and [ 13 C, 1 H]-HSQC spectra.

BTB and CNC homolog 1 (BACH1) is a master transcriptional regulator of the cellular oxidative stress response and pro-metastatic oncogene. Post-translational stability of BACH1 is tightly regulated by distinct F-box ubiquitin ligases, including SCFFBXO22 and SCFFBXL17. However, the molecular details have been elusive. Here, we reveal a structural switch in FBXO22 that controls the recognition of a three-dimensional degron in the BACH1 BTB domain, thus explaining its specificity for dimeric BACH1. We describe how cancer-associated mutations in FBXO22 modulate binding and ubiquitylation of BACH1. Further, we reveal that cancer-related mutations or cysteine-modifications destabilize the BTB domain and redirect BACH1 to FBXL17, where it is recognized as a monomer. This explains how complementary ligases post-translationally regulate BACH1 depending on the state of its BTB domain. Our findings provide mechanistic insights into the regulation of the oxidative stress response and may spur therapeutic strategies to targeting oxidative stress-related disorders and metastatic cancers.