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1.
bioRxiv ; 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38766230

RESUMO

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

2.
Cancer Res Commun ; 4(4): 986-1003, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38530197

RESUMO

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.


Assuntos
Adenosina Desaminase , Neoplasias da Mama , RNA Helicases DEAD-box , Proteínas de Neoplasias , Proteínas de Ligação a RNA , Feminino , Humanos , Neoplasias da Mama/genética , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Imunidade Inata , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA de Cadeia Dupla/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral
3.
Mol Cell ; 84(1): 107-119, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38118451

RESUMO

The ability to sense and respond to infection is essential for life. Viral infection produces double-stranded RNAs (dsRNAs) that are sensed by proteins that recognize the structure of dsRNA. This structure-based recognition of viral dsRNA allows dsRNA sensors to recognize infection by many viruses, but it comes at a cost-the dsRNA sensors cannot always distinguish between "self" and "nonself" dsRNAs. "Self" RNAs often contain dsRNA regions, and not surprisingly, mechanisms have evolved to prevent aberrant activation of dsRNA sensors by "self" RNA. Here, we review current knowledge about the life of endogenous dsRNAs in mammals-the biosynthesis and processing of dsRNAs, the proteins they encounter, and their ultimate degradation. We highlight mechanisms that evolved to prevent aberrant dsRNA sensor activation and the importance of competition in the regulation of dsRNA sensors and other dsRNA-binding proteins.


Assuntos
RNA de Cadeia Dupla , Viroses , Animais , RNA de Cadeia Dupla/genética , RNA Helicases DEAD-box/metabolismo , Imunidade Inata , Mamíferos/metabolismo
4.
bioRxiv ; 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36909617

RESUMO

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The interferon inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for ADAR1 in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110 interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.

6.
Cancer Res Commun ; 1(2): 56-64, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-35586115

RESUMO

The RNA editing enzyme ADAR, is an attractive therapeutic target for multiple cancers. Through its deaminase activity, ADAR edits adenosine to inosine in dsRNAs. Loss of ADAR in some cancer cell lines causes activation of the type I interferon pathway and the PKR translational repressor, leading to inhibition of proliferation and stimulation of cell death. As such, inhibition of ADAR function is a viable therapeutic strategy for many cancers. However, there are no FDA approved inhibitors of ADAR. Two small molecules have been previously shown to inhibit ADAR or reduce its expression: 8-azaadenosine and 8-chloroadenosine. Here we show that neither molecule is a selective inhibitor of ADAR. Both 8-azaadenosine and 8-chloroadenosine show similar toxicity to ADAR-dependent and independent cancer cell lines. Furthermore, the toxicity of both small molecules is comparable between cell lines with either knockdown or overexpression of ADAR, and cells with unperturbed ADAR expression. Treatment with neither molecule causes activation of PKR. Finally, treatment with either molecule has no effect on A-to-I editing of multiple ADAR substrates. Together these data show that 8-azaadenosine and 8-chloroadenosine are not suitable small molecules for therapies that require selective inhibition of ADAR, and neither should be used in preclinical studies as ADAR inhibitors.


Assuntos
Adenosina , Interferon Tipo I , Adenosina/farmacologia , 2-Cloroadenosina
7.
Oncogene ; 40(1): 189-202, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33110236

RESUMO

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1 knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.


Assuntos
Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
8.
Sci Rep ; 10(1): 22276, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335292

RESUMO

Tumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5'-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5'-TOP encoded proteins. The 5'-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.


Assuntos
Fator 1 de Ribosilação do ADP/genética , Neoplasias/genética , Proteínas Ribossômicas/genética , Proteína Supressora de Tumor p53/genética , Autoantígenos/genética , Proliferação de Células/genética , Fator de Iniciação Eucariótico 4G/genética , Humanos , Neoplasias/patologia , Biossíntese de Proteínas/genética , Ribonucleoproteínas/genética , Proteínas Ribossômicas/biossíntese , Ribossomos/genética , Ativação Transcricional/genética , Antígeno SS-B
9.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
10.
Nat Commun ; 9(1): 301, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29352242

RESUMO

RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3' untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the cis-acting elements of RBPs and miRNAs. We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use PTRE-seq to dissect sequence preferences and interactions between miRNAs and RBPs. The binding sites for these effector molecules influenced different aspects of the RNA lifecycle: RNA stability, translation efficiency, and translation initiation. In some cases, post-transcriptional control is modular, with different factors acting independently of each other, while in other cases factors show specific epistatic interactions. The throughput, flexibility, and reproducibility of PTRE-seq make it a valuable tool to study post-transcriptional regulation by 3'UTR elements.


Assuntos
MicroRNAs/genética , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Biblioteca Gênica , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Ligação Proteica , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA , Termodinâmica , Fatores de Transcrição/metabolismo
11.
Sci Rep ; 7(1): 14884, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097662

RESUMO

MicroRNAs are well known regulators of mRNA stability and translation. However, the magnitude of both translational repression and mRNA decay induced by miRNA binding varies greatly between miRNA targets. This can be the result of cis and trans factors that affect miRNA binding or action. We set out to address this issue by studying how various mRNA characteristics affect miRNA-mediated repression. Using a dual luciferase reporter system, we systematically analyzed the ability of selected mRNA elements to modulate miRNA-mediated repression. We found that changing the 3'UTR of a miRNA-targeted reporter modulates translational repression by affecting the translation efficiency. This 3'UTR dependent modulation can be further altered by changing the codon-optimality or 5'UTR of the luciferase reporter. We observed maximal repression with intermediate codon optimality and weak repression with very high or low codon optimality. Analysis of ribosome profiling and RNA-seq data for endogenous miRNA targets revealed translation efficiency as a key determinant of the magnitude of miRNA-mediated translational repression. Messages with high translation efficiency were more robustly repressed. Together our results reveal modulation of miRNA-mediated repression by characteristics and features of the 5'UTR, CDS and 3'UTR.


Assuntos
MicroRNAs/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Linhagem Celular , Códon/genética , Códon/metabolismo , Regulação para Baixo , Drosophila , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
12.
PLoS One ; 11(12): e0167877, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27930710

RESUMO

Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb-RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação
13.
BMC Cell Biol ; 12: 40, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21933443

RESUMO

BACKGROUND: The NAD(+)-dependent histone deacetylases, known as "sirtuins", participate in a variety of processes critical for single- and multi-cellular life. Recent studies have elucidated the importance of sirtuin activity in development, aging, and disease; yet, underlying mechanistic pathways are not well understood. Specific sirtuins influence chromatin structure and gene expression, but differences in their pathways as they relate to distinct chromatin functions are just beginning to emerge. To further define the range of global chromatin changes dependent on sirtuins, unique biological features of the ciliated protozoan Tetrahymena thermophila can be exploited. This system offers clear spatial and temporal separation of multiple whole genome restructuring events critical for the life cycle. RESULTS: Inhibition with nicotinamide revealed that sirtuin deacetylase activity in Tetrahymena cells promotes chromatin condensation during meiotic prophase, differentiation of heterochromatin from euchromatin during development, and chromatin condensation/degradation during programmed nuclear death. We identified a class I sirtuin, called Thd14, that resides in mitochondria and nucleoli during vegetative growth, and forms a large sub-nuclear aggregate in response to prolonged cell starvation that may be peripherally associated with nucleoli. During sexual conjugation and development Thd14 selectively concentrates in the parental nucleus prior to its apoptotic-like degradation. CONCLUSIONS: Sirtuin activity is important for several functionally distinct events requiring global chromatin condensation. Our findings suggest a novel role for sirtuins in promoting programmed pycnosis by acting on chromatin destined for degradation. The sirtuin Thd14, which displays physiological-dependent differential localization within the nucleus, is a candidate for a chromatin condensation enzyme that is coupled to nuclear degradation.


Assuntos
Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Sirtuínas/metabolismo , Tetrahymena/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Células Cultivadas , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Conjugação Genética/efeitos dos fármacos , Fragmentação do DNA , Meiose/efeitos dos fármacos , Dados de Sequência Molecular , Niacinamida/farmacologia , Filogenia , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética
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