RNA tailing machinery drives amyloidogenic phase transition.

The RNA tailing machinery adds nucleotides to the 3'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis.

Molecular Pathophysiology of Chronic Wounds: Current State and Future Directions.

Venous leg ulcers, diabetic foot ulcers, and pressure ulcers are complex chronic wounds with multifactorial etiologies that are associated with high patient morbidity and mortality. Despite considerable progress in deciphering the pathologies of chronic wounds using "omics" approaches, considerable gaps in knowledge remain, and current therapies are often not efficacious. We provide a comprehensive overview of current understanding of the molecular mechanisms that impair healing and current knowledge on cell-specific dysregulation including keratinocytes, fibroblasts, immune cells, endothelial cells and their contributions to impaired reepithelialization, inflammation, angiogenesis, and tissue remodeling that characterize chronic wounds. We also provide a rationale for further elucidation of ulcer-specific pathologic processes that can be therapeutically targeted to shift chronic nonhealing to acute healing wounds.

FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers.

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs.

Local translation in nuclear condensate amyloid bodies.

Biomolecular condensates concentrate molecules to facilitate basic biochemical processes, including transcription and DNA replication. While liquid-like condensates have been ascribed various functions, solid-like condensates are generally thought of as amorphous sites of protein storage. Here, we show that solid-like amyloid bodies coordinate local nuclear protein synthesis (LNPS) during stress. On stimulus, translationally active ribosomes accumulate along fiber-like assemblies that characterize amyloid bodies. Mass spectrometry analysis identified regulatory ribosomal proteins and translation factors that relocalize from the cytoplasm to amyloid bodies to sustain LNPS. These amyloidogenic compartments are enriched in newly transcribed messenger RNA by Heat Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal intergenic spacer noncoding RNA (rIGSRNA) that constructs amyloid bodies prevents recruitment of the nuclear protein synthesis machinery, abolishes LNPS, and impairs the nuclear HSF1 response. We propose that amyloid bodies support local nuclear translation during stress and that solid-like condensates can facilitate complex biochemical reactions as their liquid counterparts can.

A translational program that suppresses metabolism to shield the genome.

Translatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity. A system-wide MATRIX survey identified the ancient eIF5A as a pH-regulated translation factor that responds to fermentation-induced acidosis. TMT-pulse-SILAC analysis identified several pH-dependent proteins, including the mTORC1 suppressor Tsc2 and the longevity regulator Sirt1. Sirt1 operates as a pH-sensor that deacetylates nuclear eIF5A during anaerobiosis, enabling the cytoplasmic export of eIF5A/Tsc2 mRNA complexes for translational engagement. Tsc2 induction inhibits mTORC1 to suppress cellular metabolism and prevent acidosis-induced DNA damage. Depletion of eIF5A or Tsc2 leads to metabolic re-initiation and proliferation, but at the expense of incurring substantial DNA damage. We suggest that eIF5A operates as a translatome remodeler that suppresses metabolism to shield the genome.

A network of RNA-binding proteins controls translation efficiency to activate anaerobic metabolism.

Protein expression evolves under greater evolutionary constraint than mRNA levels, and translation efficiency represents a primary determinant of protein levels during stimuli adaptation. This raises the question as to the translatome remodelers that titrate protein output from mRNA populations. Here, we uncover a network of RNA-binding proteins (RBPs) that enhances the translation efficiency of glycolytic proteins in cells responding to oxygen deprivation. A system-wide proteomic survey of translational engagement identifies a family of oxygen-regulated RBPs that functions as a switch of glycolytic intensity. Tandem mass tag-pulse SILAC (TMT-pSILAC) and RNA sequencing reveals that each RBP controls a unique but overlapping portfolio of hypoxic responsive proteins. These RBPs collaborate with the hypoxic protein synthesis apparatus, operating as a translation efficiency checkpoint that integrates upstream mRNA signals to activate anaerobic metabolism. This system allows anoxia-resistant animals and mammalian cells to initiate anaerobic glycolysis and survive hypoxia. We suggest that an oxygen-sensitive RBP cluster controls anaerobic metabolism to confer hypoxia tolerance.

Oxygen-Sensitive Remodeling of Central Carbon Metabolism by Archaic eIF5B.

The eukaryotic translation initiation factor 5B (eIF5B) is a homolog of IF2, an ancient translation factor that enables initiator methionine-tRNAiMet (met-tRNAiMet) loading on prokaryotic ribosomes. While it can be traced back to the last universal common ancestor, eIF5B is curiously dispensable in modern aerobic yeast and mammalian cells. Here, we show that eIF5B is an essential element of the cellular hypoxic cap-dependent protein synthesis machinery. System-wide interrogation of dynamic translation machineries by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) demonstrated augmented eIF5B activity in hypoxic translating ribosomes. Global translatome studies revealed central carbon metabolism, cellular hypoxic adaptation, and ATF4-mediated stress response as major eIF5B-dependent pathways. These primordial processes rely on eIF5B even in the presence of oxygen and active eIF2, the canonical recruiter of met-tRNAiMet in eukaryotes. We suggest that aerobic eukarya retained eIF5B/IF2 to remodel anaerobic pathways during episodes of oxygen deficiency.