Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.

Dynamic Recruitment of Single RNAs to Processing Bodies Depends on RNA Functionality.

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.

Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer.

ABTRACT: Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3-8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9-11 and oncogenic12-14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.

Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response.

Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy.

Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles.

Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress response, and developmental contexts. Many important biological consequences of such "condensation" are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify, and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly.

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.

Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting.

Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.

Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.

Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP-driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre-mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP-driven cellular processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Biomolecular condensates in kidney physiology and disease.

The regulation and preservation of distinct intracellular and extracellular solute microenvironments is crucial for the maintenance of cellular homeostasis. In mammals, the kidneys control bodily salt and water homeostasis. Specifically, the urine-concentrating mechanism within the renal medulla causes fluctuations in extracellular osmolarity, which enables cells of the kidney to either conserve or eliminate water and electrolytes, depending on the balance between intake and loss. However, relatively little is known about the subcellular and molecular changes caused by such osmotic stresses. Advances have shown that many cells, including those of the kidney, rapidly (within seconds) and reversibly (within minutes) assemble membraneless, nano-to-microscale subcellular assemblies termed biomolecular condensates via the biophysical process of hyperosmotic phase separation (HOPS). Mechanistically, osmotic cell compression mediates changes in intracellular hydration, concentration and molecular crowding, rendering HOPS one of many related phase-separation phenomena. Osmotic stress causes numerous homo-multimeric proteins to condense, thereby affecting gene expression and cell survival. HOPS rapidly regulates specific cellular biochemical processes before appropriate protective or corrective action by broader stress response mechanisms can be initiated. Here, we broadly survey emerging evidence for, and the impact of, biomolecular condensates in nephrology, where initial concentration buffering by HOPS and its subsequent cellular escalation mechanisms are expected to have important implications for kidney physiology and disease.

RNA-seq of human T cells after hematopoietic stem cell transplantation identifies Linc00402 as a regulator of T cell alloimmunity.

Mechanisms governing allogeneic T cell responses after solid organ and allogeneic hematopoietic stem cell transplantation (HSCT) are incompletely understood. To identify lncRNAs that regulate human donor T cells after clinical HSCT, we performed RNA sequencing on T cells from healthy individuals and donor T cells from three different groups of HSCT recipients that differed in their degree of major histocompatibility complex (MHC) mismatch. We found that lncRNA differential expression was greatest in T cells after MHC-mismatched HSCT relative to T cells after either MHC-matched or autologous HSCT. Differential expression was validated in an independent patient cohort and in mixed lymphocyte reactions using ex vivo healthy human T cells. We identified Linc00402, an uncharacterized lncRNA, among the lncRNAs differentially expressed between the mismatched unrelated and matched unrelated donor T cells. We found that Linc00402 was conserved and exhibited an 88-fold increase in human T cells relative to all other samples in the FANTOM5 database. Linc00402 was also increased in donor T cells from patients who underwent allogeneic cardiac transplantation and in murine T cells. Linc00402 was reduced in patients who subsequently developed acute graft-versus-host disease. Linc00402 enhanced the activity of ERK1 and ERK2, increased FOS nuclear accumulation, and augmented expression of interleukin-2 and Egr-1 after T cell receptor engagement. Functionally, Linc00402 augmented the T cell proliferative response to an allogeneic stimulus but not to a nominal ovalbumin peptide antigen or polyclonal anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a regulator of allogeneic T cell function.

Antisense oligonucleotides and nucleic acids generate hypersensitive platelets.

INTRODUCTION: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia. METHODS: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo. RESULTS: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6-8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo. CONCLUSION: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment.

Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2.

Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [(35)S]guanosine 5'-(gamma-thio)triphosphate binding to Galpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.

An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development.

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.

Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.