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Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.
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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.
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From extrachromosomal DNA to neo-peptides, the broad reprogramming of the cancer genome leads to the emergence of molecules that are specific to the cancer state. We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-specific small RNAs with the potential to play functional roles in breast cancer progression1. Here, we report a systematic and comprehensive search to identify, annotate, and characterize cancer-emergent oncRNAs across 32 tumor types. We also leverage large-scale in vivo genetic screens in xenografted mice to functionally identify driver oncRNAs in multiple tumor types. We have not only discovered a large repertoire of oncRNAs, but also found that their presence and absence represent a digital molecular barcode that faithfully captures the types and subtypes of cancer. Importantly, we discovered that this molecular barcode is partially accessible from the cell-free space as some oncRNAs are secreted by cancer cells. In a large retrospective study across 192 breast cancer patients, we showed that oncRNAs can be reliably detected in the blood and that changes in the cell-free oncRNA burden captures both short-term and long-term clinical outcomes upon completion of a neoadjuvant chemotherapy regimen. Together, our findings establish oncRNAs as an emergent class of cancer-specific non-coding RNAs with potential roles in tumor progression and clinical utility in liquid biopsies and disease monitoring.
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Small non-coding RNAs can be secreted through a variety of mechanisms, including exosomal sorting, in small extracellular vesicles, and within lipoprotein complexes. However, the mechanisms that govern their sorting and secretion are not well understood. Here, we present ExoGRU, a machine learning model that predicts small RNA secretion probabilities from primary RNA sequences. We experimentally validated the performance of this model through ExoGRU-guided mutagenesis and synthetic RNA sequence analysis. Additionally, we used ExoGRU to reveal cis and trans factors that underlie small RNA secretion, including known and novel RNA-binding proteins (RBPs), e.g., YBX1, HNRNPA2B1, and RBM24. We also developed a novel technique called exoCLIP, which reveals the RNA interactome of RBPs within the cell-free space. Together, our results demonstrate the power of machine learning in revealing novel biological mechanisms. In addition to providing deeper insight into small RNA secretion, this knowledge can be leveraged in therapeutic and synthetic biology applications.
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Vesículas Extracelulares , RNA , RNA/genética , Proteínas de Ligação a RNA/genética , Vesículas Extracelulares/metabolismo , Mutagênese , Aprendizado de MáquinaRESUMO
Microsatellite instability high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion, cell proliferation, and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.
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Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis; however, its effects on cancer progression remain poorly understood. Here, to address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed dedicated regression-based methods to analyse ribosome profiling and alternative polyadenylation data, and identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as a translational controller of a specific mRNA regulon. We found that HNRNPC is downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3' untranslated region lengthening and, subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. In addition, the reduced expression of HNRNPC and its regulon is associated with the worse prognosis in breast cancer patient cohorts.
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Neoplasias da Mama , Processamento Pós-Transcricional do RNA , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
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Neoplasias da Mama , Segunda Neoplasia Primária , Neoplasias Cutâneas , Animais , Camundongos , Feminino , Humanos , Neoplasias da Mama/genética , RNA Antissenso , Quinonas/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Melanoma Maligno CutâneoRESUMO
AIMS: Diabetic ketoacidosis (DKA) is an emergency with high morbidity and mortality. This study examined patient factors associated with hospitalization for recurrent DKA. METHODS: Characteristics of 265 subjects admitted for DKA at Hennepin County Medical Center between January 2017 and January 2019 were retrospectively analyzed. Differences between subjects with a single admission versus multiple were reviewed. RESULTS: Forty-eight out of 265 patients had recurrent DKA. Risk factors included African American race (adjusted odds ratio (aOR) versus white non-Hispanic = 4.6, 95% CI 1.8-13, p = 0.001) or other race/ethnicity (aOR = 8.6, 2.9-28, p < 0.0001), younger age (aOR 37-52y versus 18-36y = 0.48, 0.19-1.16, p = 0.10; aOR 53-99y versus 18-36y = 0.37, 0.12-0.99, p = 0.05), type 1 diabetes mellitus (aOR = 2.4, 1.1-5.5, p = 0.04), ever homeless (aOR = 2.5, 1.1-5.4, p = 0.03), and drug abuse (aOR = 3.2, 1.3-7.8, p = 0.009). DKA cost a median of $29,981 per admission. CONCLUSIONS: Recurrent DKA is costly, and social determinants are strong predictors of recurrence. This study highlights the need for targeted preventative care programs.
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Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A' (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.
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Processamento Alternativo , Neoplasias da Mama/patologia , Metástase Neoplásica/genética , RNA/genética , RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Algoritmos , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Éxons , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Conformação de Ácido Nucleico , Plectina/genética , Ligação Proteica , Interferência de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , RNA-Seq , Ribonucleoproteína Nuclear Pequena U2/genética , Software , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine-tRNAGUA fragments in human cells-causing significant depletion of the precursor tRNA. Tyrosine-tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation-revealing a dedicated tRNA-regulated growth-suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.
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Códon/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Tirosina/genética , Animais , Caenorhabditis elegans/genética , Linhagem Celular , Proliferação de Células/genética , Células HEK293 , Humanos , Estresse Oxidativo/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
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.
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Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Estadiamento de Neoplasias , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , RNA-Seq , Regulon , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.
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Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
The original version of this Article contained an error in Figure 3, where panel d was inadvertently replaced with a duplicate of panel c during typesetting. Also, the legend of Figure 5f incorrectly read '310 AD patients (blue dots, r = -0.4) and 157 non-demented individuals (green dots, r = -0.1)', and should have read '310 AD patients (blue dots, r = -0.1) and 157 non-demented individuals (green dots, r = -0.4)'. Both of these errors have now been corrected in both the PDF and HTML versions of the Article.
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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.
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Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , MicroRNAs/genética , Pequeno RNA não Traduzido/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Carboxipeptidases/genética , Proliferação de Células/genética , Ácidos Nucleicos Livres/sangue , Conexinas/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , MicroRNAs/classificação , Metástase Neoplásica , Proteínas de Neoplasias/genética , Pequeno RNA não Traduzido/sangueRESUMO
INTRODUCTION: Hospitalized patients with diabetes receiving corticosteroids are at risk of developing hyperglycemia and related complications. This study evaluated a neutral protamine Hagedorn (NPH) insulin-based protocol in improving glycemic control in hospitalized patients receiving corticosteroids. METHODS: This was a randomized, prospective, non-blinded study in an inpatient setting involving patients with diabetes who were hospitalized and receiving prednisone ≥ 10 mg per day or equivalent. High dose corticosteroids group (prednisone > 40 mg/day or equivalent) received NPH insulin 0.3 U/kg between 0600 and 2000 hours if eating or 0.2 U/kg between 2000 and 0600 hours if not eating. Low dose corticosteroids group (prednisone 10-40 mg/day or equivalent) received 0.15 U/kg between 0600 and 2000 hours if eating or 0.1 U/kg between 2000 and 0600 hours if not eating. Primary outcome measure was mean blood glucose level measured pre-meal and at bedtime for days 1-5. RESULTS: Mean blood glucose level was lower in the intervention (n = 29) than in the usual care (n = 31) group [226.12 vs. 268.57 mg/dL, respectively, (95% CI for difference - 63.195 to - 21.695), p < 0.0001]. Significant differences in mean glucose level were noted at fasting [170.96 vs. 221.13 mg/dL, respectively, (95% CI for difference - 72.70 to - 27.63), p < 0.0001] and pre-lunch [208 vs. 266.48 mg/dL, respectively, (95% CI for difference - 86.61 to - 30.36), p < 0.0001]. CONCLUSION: In hospitalized patients with diabetes receiving corticosteroids, an NPH insulin-based protocol improves glycemic control. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01970241. FUNDING: Eli Lilly and Company.
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This corrects the article DOI: 10.1038/nature25465.
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Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.
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Asparagina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Animais , Asparaginase/metabolismo , Asparaginase/uso terapêutico , Asparagina/deficiência , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Disponibilidade Biológica , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Invasividade Neoplásica/patologia , Prognóstico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , Reprodutibilidade dos TestesRESUMO
The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."
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Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Modelos Biológicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Fatores de Processamento de RNA/metabolismo , Transcrição Gênica , Transcriptoma , Tristetraprolina/metabolismoRESUMO
OBJECTIVE: We discuss the implementation and outcomes of a diabetic ketoacidosis (DKA) critical care pathway (CCP) at a 462-bed teaching hospital. METHODS: A multi-disciplinary team implemented a DKA CCP that was translated into 3 computerized physician order entry (CPOE) order sets corresponding to the phases of DKA care. Historical and postintervention data were obtained via automated queries of the electronic medical record (EMR) and further analyzed by manual chart review. RESULTS: Average length of stay decreased from 104.3 to 72.9 hours (P = .0003) after implementation of a DKA CCP. CONCLUSION: Outcome data supports the use of a DKA CCP at our institution. ABBREVIATIONS: DKA = diabetic ketoacidosis CCP = critical care pathway EMR = electronic medical record CPOE = computerized physician order entry ICD-9 = International Classification of Diseases, ninth revision LoS = length of stay SQL = standard query language.
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Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3' untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)-two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes' transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization.