Long non-coding RNA lung cancer-associated transcript-1 promotes glioblastoma progression by enhancing Hypoxia-inducible factor 1 alpha activity.

BACKGROUND: Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. METHODS: lncRNAs induced by hypoxia in GSCs were identified by RNA-seq. Lung cancer-associated transcript-1 (LUCAT1) expression was assessed by qPCR, RNA-seq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, The Cancer Genome Atlas, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNA-seq, Western blot, immunohistochemistry, co-IP, ChIP, ChIP-seq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1. RESULTS: A new isoform of Lucat1 is induced by Hypoxia inducible factor 1 alpha (HIF1α) and Nuclear factor erythroid 2-related factor 2 (NRF2) in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models. CONCLUSIONS: A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.

High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p.

DEAD-box RNA helicases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box helicases unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened. Using high-resolution optical tweezers and fluorescence, we reveal a highly dynamic and stochastic process of multiple Ded1p protomers assembling on and unwinding an RNA duplex. One Ded1p protomer binds to a duplex-adjacent ssRNA tail and promotes binding and subsequent unwinding of the duplex by additional Ded1p protomers in 4-6 bp steps. The data also reveal rapid duplex unwinding and rezipping linked with binding and dissociation of individual protomers and coordinated with the ATP hydrolysis cycle.

Cellular functions of eukaryotic RNA helicases and their links to human diseases.

RNA helicases are highly conserved proteins that use nucleoside triphosphates to bind or remodel RNA, RNA-protein complexes or both. RNA helicases are classified into the DEAD-box, DEAH/RHA, Ski2-like, Upf1-like and RIG-I families, and are the largest class of enzymes active in eukaryotic RNA metabolism - virtually all aspects of gene expression and its regulation involve RNA helicases. Mutation and dysregulation of these enzymes have been linked to a multitude of diseases, including cancer and neurological disorders. In this Review, we discuss the regulation and functional mechanisms of RNA helicases and their roles in eukaryotic RNA metabolism, including in transcription regulation, pre-mRNA splicing, ribosome assembly, translation and RNA decay. We highlight intriguing models that link helicase structure, mechanisms of function (such as local strand unwinding, translocation, winching, RNA clamping and displacing RNA-binding proteins) and biological roles, including emerging connections between RNA helicases and cellular condensates formed through liquid-liquid phase separation. We also discuss associations of RNA helicases with human diseases and recent efforts towards the design of small-molecule inhibitors of these pivotal regulators of eukaryotic gene expression.

The Viral Protein K7 Inhibits Biochemical Activities and Condensate Formation by the DEAD-box Helicase DDX3X.

The DEAD-box RNA helicase DDX3X promotes translation initiation and associates with stress granules. A range of diverse viruses produce proteins that target DDX3X, including hepatitis C, dengue, vaccinia, and influenza A. The interaction of some of these viral proteins with DDX3X has been shown to affect antiviral intracellular signaling, but it is unknown whether and how viral proteins impact the biochemical activities of DDX3X and its physical roles in cells. Here we show that the protein K7 from vaccinia virus, which binds to an intrinsically disordered region in the N-terminus of DDX3X, inhibits RNA helicase and RNA-stimulated ATPase activities, as well as liquid-liquid phase separation of DDX3X in vitro. We demonstrate in HCT 116 cells that K7 inhibits association of DDX3X with stress granules, as well as the formation of aberrant granules induced by expression of DDX3X with a point mutation linked to medulloblastoma and DDX3X syndrome. The results show that targeting of the intrinsically disordered N-terminus is an effective viral strategy to modulate the biochemical functions and subcellular localization of DDX3X. Our findings also have potential therapeutic implications for diseases linked to aberrant DDX3X granule formation.

Vitamin A deficiency compromises the barrier function of the retinal pigment epithelium.

A major cause for childhood blindness worldwide is attributed to nutritional vitamin A deficiency. Surprisingly, the molecular basis of the ensuing retinal degeneration has not been well defined. Abundant expression of the retinoid transporter STRA6 in the retinal pigment epithelium (RPE) and homeostatic blood levels of retinol-binding protein delay vitamin A deprivation of the mouse eyes. Hence, genetic dissection of STRA6 makes mice susceptible to nutritional manipulation of ocular retinoid status. We performed RNA-seq analyses and complemented the data with tests of visual physiology, ocular morphology, and retinoid biochemistry to compare eyes with different vitamin A status. Mild ocular vitamin A deficiency decreased transcripts of photoreceptor transduction pathway-related genes and increased transcripts of oxidative stress pathways. The response was associated with impaired visual sensitivity and an accumulation of fluorescent debris in the retina. Severe vitamin A deficiency did not only impair visual perception but also decreased transcripts of genes encoding cell adhesion and cellular junction proteins. This response altered cell morphology, resulted in significant changes in transport pathways of small molecules, and compromised the barrier function of the RPE. Together, our analyses characterize the molecular events underlying nutritional blindness in a novel mouse model and indicate that breakdown of the outer blood-retinal barrier contributes to retinal degeneration and photoreceptor cell death in severe vitamin A deficiency.

The translation initiation factor homolog eif4e1c regulates cardiomyocyte metabolism and proliferation during heart regeneration.

The eIF4E family of translation initiation factors bind 5' methylated caps and act as the limiting step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c, for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting that Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration.

The structural basis of tRNA recognition by arginyl-tRNA-protein transferase.

Arginyl-tRNA-protein transferase 1 (ATE1) is a master regulator of protein homeostasis, stress response, cytoskeleton maintenance, and cell migration. The diverse functions of ATE1 arise from its unique enzymatic activity to covalently attach an arginine onto its protein substrates in a tRNA-dependent manner. However, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. Here, we describe the three-dimensional structures of Saccharomyces cerevisiae ATE1 with and without its tRNA cofactor. Importantly, the putative substrate binding domain of ATE1 adopts a previously uncharacterized fold that contains an atypical zinc-binding site critical for ATE1 stability and function. The unique recognition of tRNAArg by ATE1 is coordinated through interactions with the major groove of the acceptor arm of tRNA. Binding of tRNA induces conformational changes in ATE1 that helps explain the mechanism of substrate arginylation.

Two distinct binding modes provide the RNA-binding protein RbFox with extraordinary sequence specificity.

Specificity of RNA-binding proteins for target sequences varies considerably. Yet, it is not understood how certain few proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two distinct binding modes. The first exclusively accommodates cognate and closely related RNAs with high affinity. The second mode accommodates all other RNAs with reduced affinity by imposing large thermodynamic penalties on non-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA-binding site. Distinct binding modes by a single RNA-binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation in RNA-protein interactions.

Glioma stem cells activate platelets by plasma-independent thrombin production to promote glioblastoma tumorigenesis.

Background: The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example, glioblastoma (GBM) where metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcomes in many cancers. While platelets are known to promote the progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Methods: Male and female NSG mice were transplanted with GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. Results: We show that GSCs activate platelets by endogenous production of all the factors of the intrinsic and extrinsic coagulation cascades in a plasma-independent manner. Therefore, GSCs produce thrombin resulting in platelet activation. We further demonstrate that the endogenous coagulation cascades of these cancer stem cells are tumorigenic: they activate platelets to promote stemness and proliferation in vitro and pharmacological inhibition delays tumor growth in vivo. Conclusions: Our findings uncover a specific preferential relationship between platelets and GSCs that drive GBM malignancies and identify a therapeutically targetable novel interaction.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

Measuring the impact of cofactors on RNA helicase activities.

RNA helicases are the largest class of enzymes in eukaryotic RNA metabolism. In cells, protein cofactors regulate RNA helicase functions and impact biochemical helicase activities. Understanding how cofactors affect enzymatic activities of RNA helicases is thus critical for delineating physical roles and regulation of RNA helicases in cells. Here, we discuss approaches and conceptual considerations for the design of experiments to interrogate cofactor effects on RNA helicase activities in vitro. We outline the mechanistic frame for helicase reactions, discuss optimization of experimental setup and reaction parameters for measuring cofactor effects on RNA helicase activities, and provide basic guides to data analysis and interpretation. The described approaches are also instructive for determining the impact of small molecule inhibitors of RNA helicases.

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

G-quadruplex DNA inhibits unwinding activity but promotes liquid-liquid phase separation by the DEAD-box helicase Ded1p.

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid-liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains.

The kinetic landscape of an RNA-binding protein in cells.

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.

Active and Passive Destabilization of G-Quadruplex DNA by the Telomere POT1-TPP1 Complex.

Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.

Piwil1 Regulates Glioma Stem Cell Maintenance and Glioblastoma Progression.

Piwi proteins are a subfamily of Argonaute proteins that maintain germ cells in eukaryotes. However, the role of their human homologs in cancer stem cells, and more broadly in cancer, is poorly understood. Here, we report that Piwi-like family members are overexpressed in glioblastoma (GBM), with Piwil1 (Hiwi) most frequently overexpressed (88%). Piwil1 is enriched in glioma stem-like cells (GSCs) to maintain self-renewal. Silencing Piwil1 in GSCs leads to global changes in gene expression resulting in cell-cycle arrest, senescence, or apoptosis. Piwil1 knockdown increases expression of the transcriptional co-regulator BTG2 and the E3-ubiquitin ligase FBXW7, leading to reduced c-Myc expression, as well as loss of expression of stem cell factors Olig2 and Nestin. Piwil1 regulates mRNA stability of BTG2, FBXW7, and CDKN1B. In animal models of GBM, Piwil1 knockdown suppresses tumor growth and promotes mouse survival. These findings support a role of Piwil1 in GSC maintenance and glioblastoma progression.

Alternative RNA degradation pathways by the exonuclease Pop2p from Saccharomyces cerevisiae.

The 3' to 5' exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but Saccharomyces cerevisiae Pop2p harbors noncanonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S. cerevisiae Pop2p. We find that S. cerevisiae Pop2p degrades RNAs via two alternative reactions pathways, one generating nucleotides with 5'-phosphates and RNA intermediates with 3'-hydroxyls, and the other generating nucleotides with 3'-phosphates and RNA intermediates with 3'-phosphates. The enzyme is not able to initiate the reaction on RNAs with a 3'-phosphate, which leads to accumulation of RNAs with 3'-phosphates that can exceed 10 nt and are resistant to further degradation by S. cerevisiae Pop2p. We further demonstrate that S. cerevisiae Pop2p degrades RNAs in three reaction phases: an initial distributive phase, a second processive phase and a third phase during which processivity gradually declines. We also show that mutations of subsets of amino acids in the catalytic center, including those previously thought to inactivate the enzyme, moderately reduce, but not eliminate activity. Only mutation of all five amino acids in the catalytic center diminishes activity of Pop2p to background levels. Collectively, our results reveal robust exonuclease activity of S. cerevisiae Pop2p with unusual enzymatic properties, characterized by alternative degradation pathways, multiple reaction phases and functional redundancy of amino acids in the catalytic core.

Small molecules as potent biphasic modulators of protein liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.