Cushing’s syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing’s syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in approximately 45% of patients, whereas PKAcE31V, PKAcW196R and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing’s PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10ºC higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ~20 Å diameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing’s kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.
The fetal-to-adult hemoglobin transition is clinically relevant as reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and β-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1 are known post-transcriptional regulators of HbF production but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA Recognition Motif (RRM) domains and identified RBM12 as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from SCD patients with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced crosslinking and immunoprecipitation followed by high throughput sequencing (eCLIP-Seq) revealed strong preferential binding of RBM12 to 5'UTRs of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of five RRM domains as essential, and, in conjunction with a linker domain, as sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and β-thalassemia.
Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localizedin situbiotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNAXistas models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization ofXistand other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.
Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD. To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed. Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.
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