Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4

Methyl-CpG (mCpG) binding domain protein 4 (MBD4) is a member of mammalian DNA glycosylase superfamily. It contains an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain, which is an important molecule believed to be involved in maintaining of genome stability. Herein, we determined the crystal structure of C-terminal glycosylase domain of human MBD4. And the structural alignments of other helix-hairpin-helix (HhH) DNA glycosylases show that the human MBD4 glycosylase domain has the similar active site and the catalytic mechanisms as others. But the different residues in the N-terminal of domain result in the change of charge distribution on the surface of the protein, which suggest the different roles that may relate some diseases.     

蜜蜂及熊蜂微孢子虫rRNA基因研究

核糖体RNA(rRNA)是一种理想的进化计时器,已广泛地应用于研究微生物的系统发育和进化关系。蜜蜂和熊蜂都被微孢子虫感染,关于蜜蜂和熊蜂微孢子虫rRNA基因研究主要有rRNA基因序列的测定分析、二级结构的研究和用rRNA基因检测诊断微孢子虫等。这些对蜜蜂和熊蜂微孢子虫系统发育的研究和微孢子虫的防治等具有重要的推动作用。     

Efficient protein expression from the endogenous RNA polymerase I promoter using a human ribosomal DNA targeting vector

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus. Human coagulation factor IX coding sequence (FIX), with an internal ribosome entry sites element at its leader region, was targeted into the 18S rDNA locus via homologous recombination. FIX protein expression, which was under the control of the endogenous Pol I promoter, was found to be similar to that of a moderate Pol II promoter. The average FIX expression level of the rDNA recombinants was additionally enhanced to that from a strong Pol II promoter as a result of elimination of position effects. Our data suggest the possibility of applying this system in gene therapy for hereditary diseases.     

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.     

Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer

We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.     

Genetic variation in epigenetic inheritance of ribosomal RNA gene methylation in Arabidopsis

We investigated the fidelity of epigenetic inheritance in crosses between three accessions of the flowering plant Arabidopsis thaliana (Canary Islands, Cape Verde Islands, and Columbia). Specifically, we examined the cytosine methylation content of the ribosomal RNA genes at the two nucleolus organizer regions (NOR2 and NOR4) in F1 and F2 hybrid individuals derived from reciprocal crosses between the high NOR methylation strain, Columbia, and the two other accessions, both of which have less NOR methylation. In crosses between the Columbia and Cape Verde Islands strains, the cytosine methylation content segregated as an additive Mendelian trait: the high NOR methylation state was tightly associated with the inheritance of the two Columbia-derived NOR loci. First-generation hybrid individuals between the Canary Islands and Columbia strains also showed a cytosine methylation content at the NORs intermediate between the parental values, consistent with the epigenetic inheritance of parental methylation patterns. Interestingly, mapping data from F2 individuals derived from a Canary Islands x Columbia cross revealed that NOR2 accounted for nearly all of the NOR methylation variation segregating in the population. NOR4 retains a significant effect on total NOR methylation content only through a complex epistatic interaction with NOR2. Our results indicate that the inheritance of differential cytosine methylation states at NOR loci can be modified by their genetic context, opening up the possibility of genetic dissection of epigenetic inheritance.