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1.
Nucleic Acids Res ; 50(11): 6474-6496, 2022 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-35639772

RESUMEN

In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1's interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1's binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.


Asunto(s)
G-Cuádruplex , Empalme Alternativo , Secuencia de Bases , Oncogenes , ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
2.
Mol Cancer Res ; 22(7): 625-641, 2024 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-38588446

RESUMEN

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1's transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Tensinas , Humanos , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Tensinas/metabolismo , Tensinas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Sarcoma de Ewing/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Línea Celular Tumoral , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Adhesiones Focales/genética , Adhesiones Focales/metabolismo
3.
Wiley Interdiscip Rev RNA ; 14(5): e1788, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37042074

RESUMEN

The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.


Asunto(s)
Empalme del ARN , ARN , Niño , Humanos , ARN/metabolismo , Empalme Alternativo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
4.
bioRxiv ; 2023 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-38187702

RESUMEN

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma (EWS) cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in EWS cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of EWS cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. EWS cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared to control cells. The cytoskeleton of control cells and ETS1-activated EWS cell lines also differed. Specifically, control cells exhibited a distributed vinculin signal and a network-like organization of F-actin. In contrast, ETS1-activated EWS cells showed an accumulation of vinculin and F-actin towards the plasma membrane. Interestingly, the phenotype of ETS1-activated EWS cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in EWS tumors positively correlates with that of ETS1.

5.
Cancer Res ; 80(19): 4046-4057, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32616503

RESUMEN

Numerous studies have implicated changes in the Y chromosome in male cancers, yet few have investigated the biological importance of Y chromosome noncoding RNA. Here we identify a group of Y chromosome-expressed long noncoding RNA (lncRNA) that are involved in male non-small cell lung cancer (NSCLC) radiation sensitivity. Radiosensitive male NSCLC cell lines demonstrated a dose-dependent induction of linc-SPRY3-2/3/4 following irradiation, which was not observed in radioresistant male NSCLC cell lines. Cytogenetics revealed the loss of chromosome Y (LOY) in the radioresistant male NSCLC cell lines. Gain- and loss-of-function experiments indicated that linc-SPRY3-2/3/4 transcripts affect cell viability and apoptosis. Computational prediction of RNA binding proteins (RBP) motifs and UV-cross-linking and immunoprecipitation (CLIP) assays identified IGF2BP3, an RBP involved in mRNA stability, as a binding partner for linc-SPRY3-2/3/4 RNA. The presence of linc-SPRY3-2/3/4 reduced the half-life of known IGF2BP3 binding mRNA, such as the antiapoptotic HMGA2 mRNA, as well as the oncogenic c-MYC mRNA. Assessment of Y chromosome in NSCLC tissue microarrays and expression of linc-SPRY3-2/3/4 in NSCLC RNA-seq and microarray data revealed a negative correlation between the loss of the Y chromosome or linc-SPRY3-2/3/4 and overall survival. Thus, linc-SPRY3-2/3/4 expression and LOY could represent an important marker of radiotherapy in NSCLC. SIGNIFICANCE: This study describes previously unknown Y chromosome-expressed lncRNA regulators of radiation response in male NSCLC and show a correlation between loss of chromosome Y and radioresistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4046/F1.large.jpg.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Cromosomas Humanos Y/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Neoplasias Pulmonares/radioterapia , ARN Largo no Codificante/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Relación Dosis-Respuesta en la Radiación , Genes myc , Proteína HMGA2/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones Desnudos , Pronóstico , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Tolerancia a Radiación/genética , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Sci Rep ; 9(1): 3662, 2019 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-30842470

RESUMEN

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.


Asunto(s)
Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , ARN Largo no Codificante/genética , Proteínas Represoras/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/virología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Papillomavirus Humano 16/genética , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Pronóstico , Análisis de Secuencia de ARN , Análisis de Supervivencia , Regulación hacia Arriba , Neoplasias del Cuello Uterino/genética
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