RESUMEN
Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.
Asunto(s)
Células-Madre Neurales , Reparación del ADN por Recombinación , Animales , Ratones , Arginina/metabolismo , Reparación del ADN , Inestabilidad Genómica , Genómica , Histonas/genética , Histonas/metabolismo , Células-Madre Neurales/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
Leptospira interrogans (L. interrogans) genomic DNA was used as template to amplify the full-length gene for ribosomal protein L11 methyltransferase (liPrmA) by PCR. The pET22b-/liprmA expression plasmid was successfully constructed in Escherichia coli (E.coli.) strain TOP10 and confirmed by restriction enzyme digest and sequencing. Through optimizing expression of the recombinant liPrmA-6xHis fusion protein in expression host E. coli. BL21, the yield of soluble target protein reached 40 mg (liter culture)-1. The LiPrmA was purified to apparent homogeneity in a single step using Ni-NTA His Bind chromatography. Amino acid homologous analysis showed that liPrmA shared significant identity with other prokaryotic PrmA and eukaryotic putative PrmA in the catalytic region including AdoMet binding domain. Methylation activity experiments showed purified liPrmA was able to catalyze the ribosomal protein L11 of L. interrogans methylated under the presence of S-adenosyl-methionine (AdoMet).
Asunto(s)
Proteínas Bacterianas/genética , Leptospira interrogans/enzimología , Metiltransferasas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Leptospira interrogans/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Transformación GenéticaRESUMEN
The function of ver203, a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion-expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S-promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen-tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double- and single-stranded probes, and detection of GUS activity by X-gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.