RESUMEN
The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.
Asunto(s)
COVID-19 , Quinasas Janus , Animales , Citocinas/metabolismo , Humanos , Interferones/metabolismo , Quinasas Janus/metabolismo , Mamíferos/metabolismo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
By using a cell fraction technique that separates chromatin-associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown in a previous study that residues in exons are methylated (m6A) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called "epigenetic" demethylation. The turnover rate of mRNA molecules is faster, depending on m6A content in HeLa cell mRNA, suggesting that specification of mRNA stability may be the major role of m6A exon modification. In mouse embryonic stem cells (mESCs) lacking Mettl3, the major mRNA methylase, the cells continue to grow, making the same mRNAs with unchanged splicing profiles in the absence (>90%) of m6A in mRNA, suggesting no common obligatory role of m6A in splicing. All these data argue strongly against a commonly used "reversible dynamic methylation/demethylation" of mRNA, calling into question the concept of "RNA epigenetics" that parallels the well-established role of dynamic DNA epigenetics.
Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/genética , Precursores del ARN/genética , Estabilidad del ARN , ARN Mensajero/genética , Adenosina/genética , Animales , Células Madre Embrionarias , Epigénesis Genética , Exones/genética , Femenino , Células HeLa , Humanos , Metilación , Ratones , Empalme del ARNRESUMEN
Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only â¼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.
Asunto(s)
Citoplasma/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Animales , Cromatina/metabolismo , Células Madre Embrionarias , Exones/genética , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Intrones/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , RatonesRESUMEN
Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132-713) and STAT3 (127-722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.
Asunto(s)
Mutación Missense , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Activación Transcripcional , Sustitución de Aminoácidos , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Fosforilación , Multimerización de Proteína , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice.
Asunto(s)
Regiones no Traducidas 3'/genética , Adenosina/metabolismo , Metilación de ADN/genética , Exones/genética , Regulación de la Expresión Génica , ARN Mensajero/química , Animales , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Hígado/citología , Hígado/metabolismo , Ratones , Poliadenilación , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
In this Reflections, I review a few early and very lucky events that gave me a running start for the rest of a long and wonderfully enjoyable career. For the main part, a discussion is provided of what I recall as the main illuminating results that my many dozens of students and postdoctoral fellows (approximately 140 in all) provided to our biochemical/molecular biological world.
Asunto(s)
Células Eucariotas , Regulación de la Expresión Génica , Animales , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m(7)Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and â¼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about "cotranscriptional splicing." However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of â¼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3' end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm.
Asunto(s)
Biología Molecular/historia , Poli A/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Empalme del ARN , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , ARN Polimerasa II/metabolismoRESUMEN
We look back on the discoveries that the tyrosine kinases TYK2 and JAK1 and the transcription factors STAT1, STAT2, and IRF9 are required for the cellular response to type I interferons. This initial description of the JAK-STAT pathway led quickly to additional discoveries that type II interferons and many other cytokines signal through similar mechanisms. This well-understood pathway now serves as a paradigm showing how information from protein-protein contacts at the cell surface can be conveyed directly to genes in the nucleus. We also review recent work on the STAT proteins showing the importance of several different posttranslational modifications, including serine phosphorylation, acetylation, methylation, and sumoylation. These remarkably proficient proteins also provide noncanonical functions in transcriptional regulation and they also function in mitochondrial respiration and chromatin organization in ways that may not involve transcription at all.
Asunto(s)
Interferón Tipo I/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Animales , Cromatina/fisiología , Humanos , Factores Reguladores del Interferón/metabolismo , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción GenéticaAsunto(s)
Regulación de la Expresión Génica/inmunología , Subunidad alfa del Factor 3 de Genes Estimulados por el Interferón/historia , Fosfotirosina/metabolismo , Transcripción Genética/inmunología , Historia del Siglo XX , Humanos , Subunidad alfa del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Interferón gamma/historiaAsunto(s)
Transformación Celular Neoplásica/metabolismo , Glucosa/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Neoplasias/genética , Neoplasias/patología , Fosforilación , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/genéticaRESUMEN
Persistently activated STAT3 contributes to cell survival in many different human cancers. Cancer cell secretion of IL-6 is a frequent basis for persistent STAT3 activation; we show that antibodies against IL-6 or gp-130, the signaling unit of the IL-6 receptor, can abruptly remove persistently activated STAT3 causing prompt disappearance of cysteine proteases of serpin B3/B4 mRNAs, known as squamous cell carcinoma antigens 1 and 2. STAT3 occupies the promoter of serpin B3/B4 before removal and siRNA removal of B3/B4 mRNA caused cell death in HN13 head and neck cancer cells. Thus persistently activated STAT3 is a required part of the continuous activation of B3/B4 genes, which protects tumor cells from dying.
Asunto(s)
Antígenos de Neoplasias/genética , Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción STAT3/metabolismo , Serpinas/genética , Activación Transcripcional , Anticuerpos/inmunología , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Inmunoprecipitación de Cromatina , Receptor gp130 de Citocinas/análisis , Receptor gp130 de Citocinas/inmunología , Humanos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/inmunología , Regiones Promotoras GenéticasRESUMEN
The proapoptotic factors Reaper, Hid, Grim, and Sickle regulate apoptosis in Drosophila by inhibiting the antiapoptotic factor DIAP1 (Drosophila inhibitor of apoptosis 1). Heat, UV light, x-rays, and developmental signals can all increase the proapoptotic factors, but the control of transcription of the diap1 gene is unclear. We show that in imaginal discs the single Drosophila STAT protein (STAT92E) when activated can directly increase DIAP1 through binding to STAT DNA-binding sites in the diap1 promoter. The STAT92E contribution to DIAP1 production is required for cell survival after x-irradiation but not under unstressed conditions. Because DIAP1 prevents apoptosis after a variety of stresses, STAT92E may have a role in regulating stress responses in general.
Asunto(s)
Apoptosis/efectos de la radiación , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Ojo/citología , Ojo/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Factores de Transcripción STAT/deficiencia , Factores de Transcripción STAT/genéticaRESUMEN
Cooperation between STAT3 and c-Jun in driving transcription during transfection of reporter constructs is well established, and both proteins are present on some interleukin-6 (IL-6) STAT3-dependent promoters on chromosomal loci. We report that small interfering RNA knockdown of c-Jun or c-Fos diminishes IL-6 induction of some but not all STAT3-dependent mRNAs. Specific contact sites in STAT3 responsible for interaction of a domain of STAT3 with c-Jun were known. Here we show that the B-zip domain of c-Jun interacts with STAT3 and that c-Jun mutation R261A or R261D near but not in the DNA binding domain blocks in vitro STAT3-c-Jun interaction and decreases costimulation of transcription in transfection assays. Cooperative binding to DNA of tyrosine-phosphorylated STAT3 and both wild-type and R261A mutant c-Jun was observed. Even c-Jun mutant R261D, which on its own did not bind DNA, bound DNA weakly in the presence of STAT3. We conclude that a functional interaction between STAT3 and c-Jun while bound to chromosomal DNA elements exists and is necessary for driving transcription on at least some STAT3 target genes. Identifying such required interactive protein interfaces should be a stimulus to search for compounds that could ultimately inhibit the activity of STAT3 in tumors dependent on persistently active STAT3.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción AP-1/metabolismo , Secuencia de Aminoácidos , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Glutatión Transferasa/metabolismo , Humanos , Interleucina-6/farmacología , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Mutación , Plásmidos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Receptores de Interleucina-6/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/genética , Factor de Transcripción AP-1/genética , TransfecciónRESUMEN
We report experiments that infer a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, we show that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY-SH2 binding between the monomers of the dimer while ND ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation.
Asunto(s)
Modelos Moleculares , Fosfotirosina/metabolismo , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/metabolismo , Sitios de Unión , Dimerización , Humanos , Interferón gamma/metabolismo , Mutagénesis , Fosforilación , Fosfotirosina/química , Unión Proteica , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Transcripción STAT1/genética , TransfecciónAsunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Marcadores Genéticos , Quinasas Janus , Modelos Genéticos , Mutación , Proteínas Tirosina Quinasas/genética , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Factores de Transcripción/genéticaRESUMEN
Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer-derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1(-/-) and cyclin D1(-/+) fibroblasts led to anchorage-independent growth of only cyclin D1(-/+) cells. Furthermore, knockdown of cyclin D1 in breast carcinoma cells led to a reduction in anchorage-independent growth. Phosphorylation of the retinoblastoma (Rb) protein [a target of the cyclin D1/cyclin-dependent kinase 4/6 (cdk4/6) holoenzyme] was delayed in the cyclin D1(-/-) cells relative to cyclin D1(-/+) cells. The E7 oncogene, whose activity includes degradation of Rb and dissociation of Rb from E2F, did not confer anchorage-independent growth to the cyclin D1(-/-) cells but, in conjunction with vSrc, resulted in robust growth in soft agar. These results suggest both a cdk-dependent and cdk-independent role for cyclin D1 in modulating transformation by different oncogenes.