RESUMEN
The TGFß pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFß signalling could have far-reaching implications in many other cell types and in diseases such as cancer.
Asunto(s)
Adenosina/análogos & derivados , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , ARN Mensajero/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Activinas/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Ciclo Celular , Epigénesis Genética , Humanos , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteína Homeótica Nanog/metabolismo , Proteína Nodal/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/citología , Unión Proteica , Factores de Empalme de ARN , ARN Mensajero/química , ARN Mensajero/genética , Transducción de Señal , TranscriptomaRESUMEN
BACKGROUND: The quality and quantity of tumor neoantigens derived from tumor mutations determines the fate of the immune response in cancer. Frameshift mutations elicit better tumor neoantigens, especially when they are not targeted by nonsense-mediated mRNA decay (NMD). For tumor progression, malignant cells need to counteract the immune response including the silencing of immunodominant neoantigens (antigen immunoediting) and promoting an immunosuppressive tumor microenvironment. Although NMD inhibition has been reported to induce tumor immunity and increase the expression of cryptic neoantigens, the possibility that NMD activity could be modulated by immune forces operating in the tumor microenvironment as a new immunoediting mechanism has not been addressed. METHODS: We study the effect of SMG1 expression (main kinase that initiates NMD) in the survival and the nature of the tumor immune infiltration using TCGA RNAseq and scRNAseq datasets of breast, lung and pancreatic cancer. Different murine tumor models were used to corroborate the antitumor immune dependencies of NMD. We evaluate whether changes of SMG1 expression in malignant cells impact the immune response elicited by cancer immunotherapy. To determine how NMD fluctuates in malignant cells we generated a luciferase reporter system to track NMD activity in vivo under different immune conditions. Cytokine screening, in silico studies and functional assays were conducted to determine the regulation of SMG1 via IL-6/STAT3 signaling. RESULTS: IL-6/STAT3 signaling induces SMG1, which limits the expression of potent frameshift neoantigens that are under NMD control compromising the outcome of the immune response. CONCLUSION: We revealed a new neoantigen immunoediting mechanism regulated by immune forces (IL-6/STAT3 signaling) responsible for silencing otherwise potent frameshift mutation-derived neoantigens.
Asunto(s)
Mutación del Sistema de Lectura , Interleucina-6 , Humanos , Animales , Ratones , Interleucina-6/genética , Transducción de Señal , Degradación de ARNm Mediada por Codón sin Sentido , Microambiente Tumoral , Factor de Transcripción STAT3/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5' and 3' untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.
Asunto(s)
Genoma Bacteriano/genética , Procesamiento Postranscripcional del ARN/genética , ARN sin Sentido/genética , ARN Mensajero/genética , Staphylococcus aureus/genética , Transcripción Genética , Regulación Bacteriana de la Expresión Génica , Humanos , Sistemas de Lectura Abierta/genética , ARN sin Sentido/metabolismo , ARN Bacteriano/genética , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Especificidad de la EspecieRESUMEN
Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3' untranslated regions (3'UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3'UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more 'UAG' motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.