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1.
EMBO J ; 42(2): e111673, 2023 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-36514940

RESUMEN

Adenosine N6-methylation (m6A) and N6,2'-O-dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD-interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9-mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti-PD-1 antibody treatment by decreasing intratumoral TGF-ß levels and increasing intratumoral IFN-γ, TNF-α levels, and tumor-infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti-PD-1 in a context-dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS-dependent TGF-ß regulation and tumor growth. While during immunotherapy, Pcif1-Fos-TGF-ß, as well as Pcif1-Stat1/Ifitm3-IFN-γ axes, contributes to the resistance of anti-PD-1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti-PD-1 therapy, supporting that the combination of PCIF1 inhibition with anti-PD-1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)-based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof-of-concept for tumor growth suppression using LNP-CMsiRNA to silence target genes in cancer.


Asunto(s)
Neoplasias Colorrectales , Inmunoterapia , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Proteínas de la Membrana/metabolismo , Metilación , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(5): e2210361120, 2023 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-36689652

RESUMEN

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N6,2'-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.


Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2 , ARN Mensajero/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Serina Endopeptidasas
3.
Proc Natl Acad Sci U S A ; 117(33): 20159-20170, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32747553

RESUMEN

Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6 -methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.


Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Vacunas contra el Cáncer/inmunología , Lactatos/metabolismo , Melanoma/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Reguladores/fisiología , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Anticuerpos , Citocinas/genética , Citocinas/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/terapia , Metiltransferasas/genética , Metiltransferasas/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células Supresoras de Origen Mieloide/fisiología , Sitios de Empalme de ARN , Empalme del ARN , Simportadores/genética , Simportadores/metabolismo , Transcriptoma , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
4.
Nat Commun ; 12(1): 5543, 2021 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-34545078

RESUMEN

N6,2'-O-dimethyladenosine (m6Am) is an abundant RNA modification located adjacent to the 5'-end of the mRNA 7-methylguanosine (m7G) cap structure. m6A methylation on 2'-O-methylated A at the 5'-ends of mRNAs is catalyzed by the methyltransferase Phosphorylated CTD Interacting Factor 1 (PCIF1). The role of m6Am and the function of PCIF1 in regulating host-pathogens interactions are unknown. Here, we investigate the dynamics and reprogramming of the host m6Am RNA methylome during HIV infection. We show that HIV infection induces a dramatic decrease in m6Am of cellular mRNAs. By using PCIF1 depleted T cells, we identify 2237 m6Am genes and 854 are affected by HIV infection. Strikingly, we find that PCIF1 methyltransferase function restricts HIV replication. Further mechanism studies show that HIV viral protein R (Vpr) interacts with PCIF1 and induces PCIF1 ubiquitination and degradation. Among the m6Am genes, we find that PCIF1 inhibits HIV infection by enhancing a transcription factor ETS1 (ETS Proto-Oncogene 1, transcription factor) stability that binds HIV promoter to regulate viral transcription. Altogether, our study discovers the role of PCIF1 in HIV-host interactions, identifies m6Am modified genes in T cells which are affected by viral infection, and reveals how HIV regulates host RNA epitranscriptomics through PCIF1 degradation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina/análogos & derivados , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Regiones no Traducidas 5'/genética , Adenosina/metabolismo , Genoma Viral , Infecciones por VIH/virología , VIH-1/genética , Humanos , Metilación , Estabilidad Proteica , Proteolisis , Proto-Oncogenes Mas , Proteína Proto-Oncogénica c-ets-1/metabolismo , ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Transcripción Genética , Replicación Viral
5.
Cell Rep ; 35(6): 109091, 2021 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-33961823

RESUMEN

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3' end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.


Asunto(s)
COVID-19/genética , Metiltransferasas/metabolismo , SARS-CoV-2/genética , Adenosina/metabolismo , COVID-19/metabolismo , Línea Celular , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Humanos , Inmunidad Innata/genética , Metilación , Metiltransferasas/genética , ARN Viral/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , SARS-CoV-2/patogenicidad , Transducción de Señal
6.
Nat Microbiol ; 1: 16011, 2016 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-27572442

RESUMEN

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m(6)A in the immune system or its role in host-pathogen interactions. Here, we investigate the topology, dynamics and bidirectional influences of the viral-host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m(6)A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m(6)A for viral replication was demonstrated by silencing of the m(6)A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev response element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.


Asunto(s)
Adenosina/análogos & derivados , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/virología , VIH-1/crecimiento & desarrollo , Interacciones Huésped-Patógeno , ARN Mensajero/análisis , Adenosina/análisis , Línea Celular , Humanos
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