ABSTRACT
RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3' untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
ABSTRACT
Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.
Subject(s)
Blood Platelets/metabolism , Cell-Derived Microparticles/metabolism , Colonic Neoplasms/metabolism , Lung Neoplasms/metabolism , MicroRNAs/metabolism , Animals , Cell-Derived Microparticles/transplantation , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Mice , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Receptors, Proteinase-ActivatedABSTRACT
OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.
Subject(s)
8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/pharmacology , Arachidonate 12-Lipoxygenase/blood , Blood Platelets/drug effects , Chromogranins/blood , Fibrinolytic Agents/pharmacology , GTP-Binding Protein alpha Subunits, Gs/blood , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Thrombosis/prevention & control , 8,11,14-Eicosatrienoic Acid/metabolism , Animals , Arachidonate 12-Lipoxygenase/deficiency , Arachidonate 12-Lipoxygenase/genetics , Blood Platelets/metabolism , Cell Adhesion Molecules/blood , Cyclic AMP/blood , Cyclic AMP-Dependent Protein Kinases/blood , Disease Models, Animal , Fibrinolytic Agents/metabolism , Humans , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/blood , Oxidation-Reduction , Phosphoproteins/blood , Phosphorylation , Platelet Aggregation/drug effects , Shelterin Complex , Signal Transduction/drug effects , Telomere-Binding Proteins/blood , Thrombosis/blood , Thrombosis/enzymology , Thrombosis/genetics , Time FactorsABSTRACT
Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , Lung Neoplasms , Signal Transduction , Transcription Factors , YAP-Signaling Proteins , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , YAP-Signaling Proteins/metabolism , Cell Line, Tumor , Animals , Drug Resistance, Neoplasm/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Neoplasm, Residual , Mice , Focal Adhesion Kinase 1/metabolism , Focal Adhesion Kinase 1/genetics , ErbB Receptors/metabolism , ErbB Receptors/genetics , Anaplastic Lymphoma Kinase/metabolism , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Antisense RNAs are ubiquitous in human cells, yet their role is largely unexplored. Here we profiled antisense RNAs in the MDA-MB-231 breast cancer cell line and its highly lung metastatic derivative. We identified one antisense RNA that drives cancer progression by upregulating the redox enzyme NADPH quinone dehydrogenase 1 (NQO1), and named it NQO1-AS. Knockdown of either NQO1 or NQO1-AS reduced lung colonization in a mouse model, and investigation into the role of NQO1 indicated that it is broadly protective against oxidative damage and ferroptosis. Breast cancer cells in the lung are dependent on this pathway, and this dependence can be exploited therapeutically by inducing ferroptosis while inhibiting NQO1. Together, our findings establish a role for NQO1-AS in the progression of breast cancer by regulating its sense mRNA post-transcriptionally. Because breast cancer predominantly affects females, the disease models used in this study are of female origin and the results are primarily applicable to females.
Subject(s)
Breast Neoplasms , Neoplasms, Second Primary , Skin Neoplasms , Animals , Mice , Female , Humans , Breast Neoplasms/genetics , RNA, Antisense , Quinones/metabolism , NAD(P)H Dehydrogenase (Quinone)/genetics , Melanoma, Cutaneous MalignantABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by many diverse genetic etiologies. Although therapeutics that specifically target causal mutations may rescue individual types of ALS, such approaches cannot treat most patients since they have unknown genetic etiology. Thus, there is a critical need for therapeutic strategies that rescue multiple forms of ALS. Here, we combine phenotypic chemical screening on a diverse cohort of ALS patient-derived neurons with bioinformatic analysis of large chemical and genetic perturbational datasets to identify broadly effective genetic targets for ALS. We show that suppressing the gene-encoding, spliceosome-associated factor SYF2 alleviates TDP-43 aggregation and mislocalization, improves TDP-43 activity, and rescues C9ORF72 and causes sporadic ALS neuron survival. Moreover, Syf2 suppression ameliorates neurodegeneration, neuromuscular junction loss, and motor dysfunction in TDP-43 mice. Thus, suppression of spliceosome-associated factors such as SYF2 may be a broadly effective therapeutic approach for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Mice , Animals , Amyotrophic Lateral Sclerosis/genetics , Motor Neurons , Mutation , DNA-Binding Proteins/geneticsABSTRACT
Identifying master regulators that drive pathologic gene expression is a key challenge in precision oncology. Here, we have developed an analytic framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a posttranscriptional regulator of RNA stability by directly binding its target mRNAs. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients. SIGNIFICANCE: By applying a new analytic approach to transcriptomic data from clinical samples and models of colon cancer progression, we have identified RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.See related commentary by Carter, p. 1261.This article is highlighted in the In This Issue feature, p. 1241.
Subject(s)
Colonic Neoplasms/pathology , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Colon/pathology , Colonic Neoplasms/diagnosis , Colonic Neoplasms/genetics , DNA-Binding Proteins/genetics , Gene Knockdown Techniques , Humans , Intestinal Mucosa/pathology , Liver Neoplasms/secondary , Male , Mice , Neoplasm Staging , RNA Stability/genetics , RNA-Binding Proteins/genetics , RNA-Seq , Regulon , Xenograft Model Antitumor AssaysABSTRACT
Here we performed a systematic search to identify breast-cancer-specific small noncoding RNAs, which we have collectively termed orphan noncoding RNAs (oncRNAs). We subsequently discovered that one of these oncRNAs, which originates from the 3' end of TERC, acts as a regulator of gene expression and is a robust promoter of breast cancer metastasis. This oncRNA, which we have named T3p, exerts its prometastatic effects by acting as an inhibitor of RISC complex activity and increasing the expression of the prometastatic genes NUPR1 and PANX2. Furthermore, we have shown that oncRNAs are present in cancer-cell-derived extracellular vesicles, raising the possibility that these circulating oncRNAs may also have a role in non-cell autonomous disease pathogenesis. Additionally, these circulating oncRNAs present a novel avenue for cancer fingerprinting using liquid biopsies.
Subject(s)
Breast Neoplasms/genetics , Cell-Free Nucleic Acids/genetics , MicroRNAs/genetics , RNA, Small Untranslated/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Breast Neoplasms/blood , Breast Neoplasms/pathology , Carboxypeptidases/genetics , Cell Proliferation/genetics , Cell-Free Nucleic Acids/blood , Connexins/genetics , Disease Progression , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , MCF-7 Cells , MicroRNAs/classification , Neoplasm Metastasis , Neoplasm Proteins/genetics , RNA, Small Untranslated/bloodABSTRACT
Due to an increasing diversity of bacterial pathogens known to be transmitted by hard ticks (Acari: Ixodidae) in North America, a comprehensive assay is needed to detect and differentiate among these numerous tick-borne pathogens. We describe an expanded protocol using a combination of multiplex polymerase chain reaction and reverse line blot hybridization to detect a greater diversity of infectious agents than were previously detectable. Ten novel oligonucleotide probes, either individually or in concert, enabled or enhanced identification of six Borrelia species, three Rickettsia species, and one Ehrlichia species. Simultaneous detection of these numerous tick-borne pathogens can advance surveillance efforts and improve accuracy of detection and, thus, reporting.
Subject(s)
Arachnid Vectors/microbiology , Borrelia/isolation & purification , Ehrlichia/isolation & purification , In Situ Hybridization/methods , Rickettsia/isolation & purification , Tick-Borne Diseases/microbiology , Ticks/microbiology , Animals , Arachnid Vectors/physiology , Borrelia/genetics , Ehrlichia/genetics , Multiplex Polymerase Chain Reaction , North America , Rickettsia/genetics , Tick-Borne Diseases/transmission , Ticks/physiologyABSTRACT
The market and popularity of e-cigarettes are growing rapidly worldwide.However,the research and regulation of this novel product have lagged behind.In this paper,we review and compare the regulatory mechanisms regarding e-cigarettes among ten Association of Southeast Asian Nations (ASEAN) member states.Our findings demonstrate a continuum of the legal regulations of e-cigarettes in Southeast Asia:on the one end,countries such as Brunei,Cambodia,Thailand,and Singapore have prohibited the importation and sale of e-cigarettes with existing tobacco control laws or specific regulations;on the other end,countries such as Myanmar and Vietnam have almost no legal restrictions on e-cigarettes whatsoever.This overview of national regulations will not only clarify the legal status of e-cigarettes in Southeast Asia but also provide some informing lessons for the tobacco control efforts in this region.