Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined ‘functional hotspots’. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

AbstractTargeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined “functional hotspots”. Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms.

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Here, we report the design and synthesis of a first macrocyclic PROTAC by adding a second cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second Brd4 bromodomain validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.

Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e., fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-l-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy, and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel–Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia-inducible factor 1 alpha (HIF-1α) substrate peptides and peptidomimetic ligands that form part of PROTAC (proteolysis targeting chimera) conjugates for targeted protein degradation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organic chemists, chemical biologists, and drug discoverers alike.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY proteins.

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied duringCaenorhabditis elegansgermline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 inC. elegansaffects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found thatnhl-2andvig-1, two known modulators of miRNA function, genetically interact with GLD-1.gld-1mutations enhance multiple phenotypes conferred bymir-35andlet-7family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred bylet-7andgld-1during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed inlet-7andgld-1mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.

Pluripotent cells have the unique ability to differentiate into diverse cell types. Over the past decade our understanding of the mechanisms underlying pluripotency, and particularly the role of transcriptional regulation, has increased dramatically. However, there is growing evidence for 'RNA-based' regulation of pluripotency. We use this term to describe control of gene expression by RNA-binding proteins (RBPs) and regulatory non-coding RNAs (ncRNAs). These molecules bind to specific elements within mRNAs and, by recruiting various effectors, affect many aspects of mRNA regulation. Here, we discuss the role of RBPs and ncRNAs in both the induction and maintenance of pluripotency. We highlight and contrast examples from pluripotent cell lines and in vivo systems while discussing the connection to transcriptional regulators.

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are "stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1-dependent repression prevents precocious translation of OET-promoting mRNAs. Secondly, GLD-1- and CGH-1-dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised.

RNA‐binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP‐mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD‐1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD‐1 interactions with the transcriptome, we identified GLD‐1‐associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD‐1 association with mRNA is determined by the strength and number of 7‐mer GLD‐1‐binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD‐1/GBM‐binding experiments to determine relative affinity, and in vivo, by ‘transplantation’ experiments, where ‘weak’ and ‘strong’ GBMs imposed translational repression of increasing strength on a non‐target mRNA. This study demonstrates that transcriptome‐wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post‐transcriptional regulatory networks. The Caenorhabditis elegans RNA‐binding protein GLD‐1 is a critical regulator of germline fate. Here, a quantitative approach to identifying GLD‐1 target RNAs provides insight into the post‐transcriptional regulatory networks underlying germ cell development.

Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, we demonstrate that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. We describe a mechanism that prevents precocious mitosis in germ cells undergoing meiosis, propose that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and provide a paradigm for the possible origin of human teratomas.

Genes can maintain spatiotemporal expression patterns by long-range interactions between cis-acting elements. The cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed primarily in epithelial cells. An element located within a DNase I-hypersensitive site (DHS) 10 kb into the first intron was previously shown to augment CFTR promoter activity in a tissue-specific manner. Here, we reveal the mechanism by which this element influences CFTR transcription. We employed a high-resolution method of mapping DHS using tiled microarrays to accurately locate the intron 1 DHS. Transfection of promoter-reporter constructs demonstrated that the element displays classical tissue-specific enhancer properties and can independently recruit factors necessary for transcription initiation. In vitro DNase I footprinting analysis identified a protected region that corresponds to a conserved, predicted binding site for hepatocyte nuclear factor 1 (HNF1). We demonstrate by electromobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) that HNF1 binds to this element both in vitro and in vivo. Moreover, using chromosome conformation capture (3C) analysis, we show that this element interacts with the CFTR promoter in CFTR-expressing cells. These data provide the first insight into the three- dimensional (3D) structure of the CFTR locus and confirm the contribution of intronic cis-acting elements to the regulation of CFTR gene expression.

Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.

Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.