RESUMEN
The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.
Asunto(s)
Glutamina , Ácidos Cetoglutáricos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/metabolismo , Linfocitos T CD4-Positivos/metabolismoRESUMEN
Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.
Asunto(s)
Edición Génica/métodos , ARN Guía de Kinetoplastida/metabolismo , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Humanos , Mutagénesis/genética , Mutagénesis/fisiología , ARN Guía de Kinetoplastida/genéticaRESUMEN
Influenza virus outbreaks remain a serious threat to public health. A greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo-infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus-infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes compared to AEC from uninfected animals, directly infected cells produce more interferon and express lower levels of Wnt signaling-associated transcripts, while concurrently expressing more transcripts associated with cell death pathways, than bystander uninfected AEC. The Wnt signaling pathway was downregulated in both in vivo-infected AEC and in vitro-infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein-expressing influenza virus, we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis, as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.
Asunto(s)
Células Epiteliales Alveolares/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Mucosa Respiratoria/inmunología , Vía de Señalización Wnt/inmunología , Células A549 , Células Epiteliales Alveolares/virología , Animales , Línea Celular , Perros , Femenino , Perfilación de la Expresión Génica , Humanos , Interferón Tipo I/inmunología , Interferones/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/virología , Mucosa Respiratoria/citología , Mucosa Respiratoria/virología , Transcriptoma/genética , Vía de Señalización Wnt/genética , Interferón lambdaRESUMEN
Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-µ at the pre-BCR checkpoint.
Asunto(s)
Linfocitos B/citología , Proteínas Nucleares/fisiología , Proteínas de Unión al ARN/fisiología , Fase S/fisiología , Tristetraprolina/fisiología , Animales , Factor 1 de Respuesta al Butirato , Secuencia Conservada , Ciclinas/metabolismo , Fase G1/genética , Fase G1/fisiología , Regulación de la Expresión Génica , Cadenas mu de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Receptores de Células Precursoras de Linfocitos B , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/fisiología , Fase S/genética , Selección Genética , Transcripción Genética , Tristetraprolina/genética , Recombinación V(D)JRESUMEN
The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specific E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro, and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the first E2 enzyme but also the first known substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans.
Asunto(s)
Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Células HEK293 , Humanos , Unión Proteica/genética , Unión Proteica/fisiología , ARN Interferente Pequeño , Proteínas SNARE , Técnicas del Sistema de Dos Híbridos , Ubiquitina/genética , Ubiquitinas/genética , Proteínas de Transporte VesicularRESUMEN
Due to the fact that mutations and up- or downregulation of genes can lead to the development of cancer, quantitative comparison of relative gene expression in healthy and cancerous tissue can gain valuable insights into tumorigenesis. While the semi-quantitative DNA microarrays are being used to identify differentially expressed genes on a genomic scale, real-time RT-PCR provides a powerful tool for quantitative measurement of gene expression. Presently, it is the most sensitive method available. Here we describe in detail a SYBR GreenI-based assay using the LightCycler instrument to measure the levels of mRNA for the ubiquitin-like protein FAT10 relative to 18S rRNA in human hepatocellular carcinoma tissue. This method can be easily adapted to any tissue (human or mouse, rat, etc.) and any gene.
Asunto(s)
Carcinoma Hepatocelular/genética , Expresión Génica , Neoplasias Hepáticas/genética , ARN Mensajero/análisis , ARN Ribosómico 18S/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ubiquitinas/genética , Benzotiazoles , Diaminas , Humanos , Compuestos Orgánicos , QuinolinasRESUMEN
Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.