RESUMEN
Interferon-stimulated genes (ISGs) comprise a program of immune effectors important for host immune defense. When uncontrolled, ISGs play a central role in interferonopathies and other inflammatory diseases. The mechanisms responsible for turning on ISGs are not completely known. By investigating MATRIN3 (MATR3), a nuclear RNA-binding protein mutated in familial ALS, we found that perturbing MATR3 results in elevated expression of ISGs. Using an integrative approach, we elucidate a pathway that leads to activation of cGAS-STING. This outlines a plausible mechanism for pathogenesis in a subset of ALS, and suggests new diagnostic and therapeutic approaches for this fatal disease.
RESUMEN
Human GW182 family proteins have Argonaute (AGO)-binding domains in their N-terminal regions and silencing domains, which interact with RNA silencing-related proteins, in their C-terminal regions. Thus, they function as scaffold proteins between the AGO protein and RNA silencing-related proteins, such as carbon catabolite repressor4-negative on TATA (CCR4-NOT) or poly(A)-binding protein (PABP). Our mass spectrometry analysis and the phosphorylation data registered in PhosphoSitePlus, a post-translational modification database, suggested that the C-terminal region of a human GW182 family protein, TNRC6A, has at least four possible phosphorylation sites, which are located near the region interacting with the CCR4-NOT complex. Among them, two serine residues at amino acid positions 1332 and 1346 (S1332 and S1346) were certainly phosphorylated in human HeLa cells, but other two serine residues (S1616 and S1691) were not phosphorylated. Furthermore, it was revealed that the phosphorylation patterns of TNRC6A affect the interaction with the CCR4-NOT complex. When S1332 and S1346 were dephosphorylated, the interactions of TNRC6A with the CCR4-NOT complex were enhanced, and when S1616 and S1691 were phosphorylated, such interaction was suppressed. Thus, phosphorylation of TNRC6A was considered to regulate the interaction with RNA silencing-related factors that may affect RNA silencing activity.
Asunto(s)
Autoantígenos/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Proteínas de Unión al ARN/genética , Receptores CCR4/genética , Aminoácidos/genética , Proteínas Argonautas/genética , Núcleo Celular/genética , Células HeLa , Humanos , MicroARNs/genética , Complejos Multiproteicos/genética , Fosforilación/genética , Interferencia de ARNRESUMEN
Trinucleotide repeat-containing gene 6A protein (TNRC6A) is an essential protein for microRNA-mediated gene silencing. TNRC6A functions in the cytoplasm as a platform protein interacting with Argonaute protein, on which microRNA is loaded for RNA silencing, and decapping enzymes or deadenylation protein complexes to induce mRNA degradation. We previously revealed that TNRC6A shuttles between the cytoplasm and nucleus. However, the function of TNRC6A in the nucleus is unclear. Here, we performed a comprehensive identification of the nuclear and cytoplasmic interacting proteins of TNRC6A protein by mass spectrometry and identified multiple proteins involved in the nuclear and cytoplasmic complexes. We found that many RNA degradation pathway proteins were involved in both nuclear and cytoplasmic TNRC6A complexes, suggesting that RNA silencing may occur via TNRC6A in both nucleus and cytoplasm or that they were involved in other important function in the nucleus. Furthermore, proteins identified in the nuclear TNRC6A complex were categorized into the spliceosomal pathway. This may mean that TNRC6A regulates splicing in the nucleus. In contrast, pathogen infection- and RNA transport-associated proteins were identified in the cytoplasmic TNRC6A complex. Thus, TNRC6A may be also involved in these pathways in the cytoplasm.
Asunto(s)
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ARN/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de MasasRESUMEN
GW182 family proteins play important roles in microRNA (miRNA)-mediated RNA silencing. They directly interact with Argonaute (Ago) proteins in processing bodies (P bodies), cytoplasmic foci involved in mRNA degradation and storage. Recently, we revealed that a human GW182 family protein, TNRC6A, is a nuclear-cytoplasmic shuttling protein, and its subcellular localization is regulated by its own nuclear localization signal and nuclear export signal. Regarding the further controlling mechanism of TNRC6A subcellular localization, we found that TNRC6A protein is tethered in P bodies by direct interaction with Ago2 under Ago2 overexpression condition in HeLa cells. Furthermore, it was revealed that such Ago proteins might be strongly tethered in the P bodies through Ago-bound small RNAs. Thus, our results indicate that TNRC6A subcellular localization is substantially controlled by the interaction with Ago proteins. Furthermore, it was also revealed that the TNRC6A subcellular localization affects the RNA silencing activity.