An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs

Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.     

Ctf4p facilitates Mcm10p to promote DNA replication in budding yeast

Ctf4p (chromosome transmission fidelity) has been reported to function in DNA metabolism and sister chromatid cohesion in Saccharomyces cerevisiae. In this study, a ctf4^S143F mutant was isolated from a yeast genetic screen to identify replication-initiation proteins. The ctf4^S143F mutant exhibits plasmid maintenance defects which can be suppressed by the addition of multiple origins to the plasmid, like other known replication-initiation mutants. We show that both ctf4^S143F and ctf4Δ strains have defects in S phase entry and S phase progression at the restrictive temperature of 38 °C. Ctf4p localizes in the nucleus throughout the cell cycle but only starts to bind chromatin at the G1/S transition and then disassociates from chromatin after DNA replication. Furthermore, Ctf4p interacts with Mcm10p physically and genetically, and the chromatin association of Ctf4p depends on Mcm10p. Finally, deletion of CTF4 destabilizes Mcm10p and Pol α in both mcm10-1 and MCM10 cells. These data indicate that Ctf4p facilitates Mcm10p to promote the DNA replication.     

RNA干扰分子的制作

小干扰RNA是一种能够在各种生物体和细胞(包括蠕虫、果蝇、植物、哺乳动物)中减弱基因表达的有效工具。在哺乳动物中转染的siRNA能够抑制特殊基因的表达,这已经证明是探索基因功能、基因敲除、抗病毒研究、基因治疗的有效方法。简单、有效、特异性地抑制基因的表达具有巨大的科学、商业和医学治疗价值。如何设计和制作siRNA是影响RNA干扰效率的一个很重要的方面。本文就siRNA的设计和制作等方面作扼要的介绍。     

The Shuttling Protein Npl3 Promotes Translation Termination Accuracy in Saccharomyces cerevisiae

Heterogeneous nuclear ribonucleoproteins are multifunctional proteins that bind to newly synthesized mRNAs in the nucleus and participate in many subsequent steps of gene expression. A well-studied Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein that has several nuclear functions is Npl3p. Here, we provide evidence that Npl3p also has a cytoplasmic role: it functions in translation termination fidelity. Yeast harboring the npl3-95 mutant allele have an impaired ability to translate lacZ, enhanced sensitivity to cycloheximide and paromomycin, and increased ability to read through translation termination codons. Most of these defects are enhanced in yeast that also lack Upf1p, an RNA surveillance factor crucial for translation termination. We show that the npl3-95 mutant allele encodes a form of Npl3p that is part of high molecular-weight complexes that cofractionate with the poly(A)-binding protein Pab1p. Together, these results lead us to propose a model in which Npl3p engenders translational fidelity by promoting the remodeling of mRNPs during translation termination.     

The Exonuclease Activity of the Yeast Mitochondrial DNA Polymerase γ Suppresses Mitochondrial DNA Deletions Between Short Direct Repeats in Saccharomyces cerevisiae

The importance of mitochondrial DNA (mtDNA) deletions in the progeroid phenotype of exonuclease-deficient DNA polymerase γ mice has been intensely debated. We show that disruption of Mip1 exonuclease activity increases mtDNA deletions 160-fold, whereas disease-associated polymerase variants were mostly unaffected, suggesting that exonuclease activity is vital to avoid deletions during mtDNA replication.     

The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum

Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼ 35-kb circular DNA genome of unusually high A/T content (> 86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3′ → 5′ exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T → C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.