RESUMEN
Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.
Asunto(s)
Macrófagos Peritoneales/inmunología , Degradación de ARNm Mediada por Codón sin Sentido/inmunología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Ribonucleasas/genética , Transactivadores/genética , Animales , Fibroblastos/citología , Fibroblastos/inmunología , Células HEK293 , Células HeLa , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inmunidad Innata , Inflamación , Secuencias Invertidas Repetidas , Macrófagos/citología , Macrófagos/inmunología , Macrófagos Peritoneales/citología , Ratones , Ratones Noqueados , Mutación , Cultivo Primario de Células , Unión Proteica , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/inmunología , ARN Mensajero/metabolismo , Ribonucleasas/deficiencia , Ribonucleasas/inmunología , Imagen Individual de Molécula , Transactivadores/inmunologíaRESUMEN
The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.
Asunto(s)
Linfocitos B , Diferenciación Celular , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas , Animales , Linfocitos B/metabolismo , Linfocitos B/citología , Ratones , ARN Helicasas/metabolismo , ARN Helicasas/genética , Ratones Noqueados , Ratones Endogámicos C57BL , Transactivadores/metabolismo , Transactivadores/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Respuesta de Proteína Desplegada/genética , Humanos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genéticaRESUMEN
Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1ß- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1ß or TLR stimulus dynamically induced the formation of Regnase-1-ß-transducin repeat-containing protein (ßTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by ßTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-ßTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from ßTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/genética , Complejos Multiproteicos/genética , Estabilidad del ARN/genética , ARN Mensajero/química , Ribonucleasas/genética , Animales , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Ribonucleasas/metabolismoRESUMEN
RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.