A unified mechanism for intron and exon definition and back-splicing.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice

The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface

In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Two-component systems generally consist of a sensor histidine kinase and a response regulator, which work together through histidyl-aspartyl phosphorelay to result in gene regulation. One of the two-component systems in Escherichia coli, CusS-CusR, is known to induce expression of cusCFBA genes at increased periplasmic Cu(I) and Ag(I) concentrations to help maintain metal ion homeostasis. CusS is a membrane-associated histidine kinase with a periplasmic sensor domain connected to the cytoplasmic ATP binding and catalytic domains through two transmembrane helices. The mechanism of how CusS senses increasing metal ion concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. The structure reveals that CusS forms a homodimer with four Ag(I) binding sites per dimeric complex. Two symmetric metal binding sites are found at the dimeric interface, which are each formed by two histidines and one phenylalanine with an unusual cation-π interaction. The other metal ion binding sites are in a nonconserved region within each monomer. Functional analyses of CusS variants with mutations in the metal sites suggest that the metal ion binding site at the dimer interface is more important for function. The structural and functional data provide support for a model in which metal-induced dimerization results in increases in kinase activity in the cytoplasmic domains of CusS

Mass spectrometry-based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the C region of fibrinogen . Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution

The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface

In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Two-component systems generally consist of a sensor histidine kinase and a response regulator, which work together through histidyl-aspartyl phosphorelay to result in gene regulation. One of the two-component systems in <i>Escherichia coli</i>, CusS–CusR, is known to induce expression of <i>cusCFBA</i> genes at increased periplasmic Cu­(I) and Ag­(I) concentrations to help maintain metal ion homeostasis. CusS is a membrane-associated histidine kinase with a periplasmic sensor domain connected to the cytoplasmic ATP binding and catalytic domains through two transmembrane helices. The mechanism of how CusS senses increasing metal ion concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag­(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. The structure reveals that CusS forms a homodimer with four Ag­(I) binding sites per dimeric complex. Two symmetric metal binding sites are found at the dimeric interface, which are each formed by two histidines and one phenylalanine with an unusual cation−π interaction. The other metal ion binding sites are in a nonconserved region within each monomer. Functional analyses of CusS variants with mutations in the metal sites suggest that the metal ion binding site at the dimer interface is more important for function. The structural and functional data provide support for a model in which metal-induced dimerization results in increases in kinase activity in the cytoplasmic domains of CusS

A unified mechanism for intron and exon definition and back-splicing.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes

Blood donor exposome and impact of common drugs on red blood cell metabolism.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality

Blood donor exposome and impact of common drugs on red blood cell metabolism

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures (“exposome”) may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell–Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine — though rarely detected in the blood donor population — has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could — through mechanisms involving sphingosine 1–phosphate–dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin — improve erythrocyte metabolism and storage quality