RESUMEN
Cytosine DNA methylation is an epigenetic mark frequently associated with silencing of genes and transposons. In Arabidopsis, the establishment of cytosine DNA methylation is performed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). DRM2 is guided to target sequences by small interfering RNAs (siRNAs) in a pathway termed RNA-directed DNA methylation (RdDM). We performed a screen for mutants that affect the establishment of DNA methylation by investigating genes that contain predicted RNA-interacting domains. After transforming FWA into 429 T-DNA insertion lines, we assayed for mutants that exhibited a late-flowering phenotype due to hypomethylated, thus ectopically expressed, copies of FWA. A T-DNA insertion line within the coding region of the spliceosome gene SR45 (sr45-1) flowered late after FWA transformation. Additionally, sr45-1 mutants display defects in the maintenance of DNA methylation. DNA methylation establishment and maintenance defects present in sr45-1 mutants are enhanced in dcl3-1 mutant background, suggesting a synergistic cooperation between SR45 and DICER-LIKE3 (DCL3) in the RdDM pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Metilación de ADN , Empalme del ARN , ARN de Planta/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , MutaciónRESUMEN
T cell receptor (TCR) signaling enhances beta1,6GlcNAc-branching in N-glycans, a phenotype that promotes growth arrest and inhibits autoimmunity by increasing surface retention of cytotoxic T lymphocyte antigen-4 (CTLA-4) via interactions with galectins. N-Acetylglucosaminyltransferase V (MGAT5) mediates beta1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi alpha1,2-mannosidase I (MIa,b,c), MGAT1, alpha1,2-mannosidase II (MII, IIx), and MGAT2. Here we report that TCR signaling enhances mRNA levels of MIa,b,c and MII,IIx in parallel with MGAT5, whereas limiting levels of MGAT1 and MGAT2. Blocking the increase in MI or MII enzyme activity induced by TCR signaling with deoxymannojirimycin or swainsonine, respectively, limits beta1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an approximately 250- and approximately 20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits beta1,6GlcNAc branching by limiting UDP-GlcNAc supply to MGAT5, suggesting that restricted changes in MGAT1 and MGAT2 mRNA levels in TCR-stimulated cells serves to enhance availability of UDP-GlcNAc to MGAT5. Together, these data suggest that TCR signaling differentially regulates multiple N-glycan-processing enzymes at the mRNA level to cooperatively promote beta1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance.
Asunto(s)
Aparato de Golgi/metabolismo , Polisacáridos/química , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Antígenos CD/metabolismo , Antígeno CTLA-4 , Proliferación Celular , Humanos , Células Jurkat , Ratones , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.
Asunto(s)
Arabidopsis/genética , Nucléolo Celular/genética , Cromatina/genética , ADN de Plantas/genética , ARN Interferente Pequeño/genética , ARN/biosíntesis , ARN/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Metilación de ADN , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Silenciador del Gen/fisiología , Sustancias Macromoleculares/metabolismo , Mutación/genética , Plantas Modificadas Genéticamente , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transducción de Señal/genéticaRESUMEN
ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.