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Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. While cellular studies indicate heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription, fluorescence-based biochemical assays and crystal structures suggest that heme displaces NCoR. Here, we show that heme artifactually influences detection of NCoR interaction in fluorescence-based assays. However, using fluorescence-independent methods, isothermal titration calorimetry and NMR spectroscopy, we demonstrate that heme directly increases REV-ERBβ ligand-binding domain (LBD) binding affinity for NCoR. We further report two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, which reveal the structural basis for heme-dependent NCoR binding to REV-ERBβ. By resolving previous contradictory biochemical, structural, and cellular studies, our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16–E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16–E21) and cochlear ganglia (E19–P3), and also at moderate levels in retina (E18–E19) and spleen (E21–P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders.

In eukaryotes, mRNA polyadenylation is a well-known modification that is essential for many aspects of the protein-coding RNAs life cycle. However, modification of the 3′ terminal nucleotide within various RNA molecules is a general and conserved process that broadly modulates RNA function in all kingdoms of life. Numerous types of modifications have been characterized, which are generally specific for a given type of RNA such as the CCA addition found in tRNAs. In recent years, the addition of nontemplated uridine nucleotides or uridylation has been shown to occur in various types of RNA molecules and in various cellular compartments with significantly different outcomes. Indeed, uridylation is able to alter RNA half-life both in positive and in negative ways, highlighting the importance of the enzymes in charge of performing this modification. The present review aims at summarizing the current knowledge on the various processes leading to RNA 3′-end uridylation and on their potential impacts in various diseases.

Nous avons étudié les aspects structuraux et fonctionnels de deux poly(U) polymérases (PUPs) afin de mieux comprendre le fonctionnement de ces enzymes: l'enzyme Cid1 PUP de Schizosaccharomyces pombe et l'enzyme RET1 de Trypanosome brucei. Ces protéines sont impliquées dans la polyuridylation de plusieurs types d'ARNs comme les ARNms ou les ARNgs. Nous avons déterminé la structure atomique par cristallographie aux rayons X de l'enzyme Cid1 liée à son substrat UTP, un UTP non hydrolysable et un pseudo-produit ApU qui correspondrait à la première étape de l'uridylation d'un ARNm polyadenylé. Récemment, nous avons obtenu des cristaux contenant un fragment fonctionnel de la protéine RET1 et la résolution de la structure est la prochaine étape. Nos études fournissent une vision atomique de la sélectivité envers l'UTP ainsi que la reconnaissance de l'ARN par les enzymes impliquées dans la polyuridylation en détaillant les mécanismes moléculaires de cette famille d'enzymes et de leurs complexes.

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to cancer tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state, with the KH12 domains of each IMP2 molecule forming the dimer interface. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function with more effective IMP2 inhibitors., Competing Interests: DECLARATION OF INTERESTS M.J. is a member of a scientific advisory board at ResistanceBio.