In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial ...cellular fate changes, cell differentiation, or malignant transformation are now known to involve ‘metabolic remodeling’ with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation (‘moonlighting’) and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme–RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here, we introduce XRNAX, ...a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs) and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-related changes in the total proteome. In addition, we combine XRNAX with crosslinking immunoprecipitation sequencing (CLIP-seq) to validate the interaction of ncRNA with lamin B1 and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.
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•XRNAX purifies protein-crosslinked RNA of all biotypes from UV-crosslinked cells•Discovery of the WKF RNA-binding domain•Discovery of more than 700 proteins interacting with non-polyadenylated RNA•Profiling of stress-induced changes in RNA-binding proteomes
A general approach for characterizing cellular RNA-protein interactions allows examination of dynamic changes to the RNA-bound proteome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native ...RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells.
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•Experimental generation of an atlas of RNA-binding sites (RBS) in human cells•RBS overlap with enzymatic cores and protein-protein interaction sites•About half of the total RBS map to disordered protein regions•RBS are enriched for phosphorylation, acetylation, and methylation sites
Many recently discovered RNA-binding proteins (RBPs) do not show architectural similarities with classical RBPs, and their modes of interaction with RNA were unclear. We developed and employed RBDmap as a method for the comprehensive determination of the RNA-interacting sites of RBPs, identifying more than a thousand such sites. These data yield unprecedented insight into RNA-protein interactions in cells with implications for numerous biological contexts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and ...comprehensive approach, termed “interactome capture,” to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
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► Interactome capture identifies hundreds of RBPs previously unknown to bind RNA ► Some globular and disordered regions represent unorthodox binding architectures ► Many RNA binders are linked to Mendelian disease ► Metabolic enzymes moonlighting as RBPs
Interactome capture identifies the landscape of mRNA-binding proteins in mammalian cells, revealing new roles for metabolic enzymes and a propensity for globular and unstructured regions to associate with messages.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Aims
Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE−/− FPNwt/C326S), ...which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI).
Methods and results
Compared with normo-ferremic ApoE−/− mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE−/− FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE−/− FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation.
Conclusion
Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.
Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA ...interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.
NMD (nonsense-mediated mRNA decay) belongs to the best-studied mRNA surveillance systems of the cell, limiting the synthesis of truncated and potentially harmful proteins on the one hand and playing ...an initially unexpected role in the regulation of global gene expression on the other hand. In the present review, we briefly discuss the factors involved in NMD, the different models proposed for the recognition of PTCs (premature termination codons), the diverse physiological roles of NMD, the involvement of this surveillance pathway in disease and the current strategies for medical treatment of PTC-related diseases.
Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain ...unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
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•Ded1p phase-separates in response to heat and pH to form gel condensates•Condensation inactivates Ded1p and represses housekeeping mRNAs•Ded1p condensation promotes stress protein production and limits cell growth•Ded1p condensation is adapted to the maximum growth temperature of a species
Heat-induced phase separation of a helicase promotes a switch in translation from housekeeping transcripts to stress-response transcripts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background & Aims The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic ...interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP–SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes. Methods We explore protein–protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells. Results Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120–139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS. Conclusions Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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