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ABSTRACT: RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. In this study, two functional nuclear exporting signals (NESs; pNES2 and pNES3) were identified at the C-terminus of RecQL4. Deletion of pNES2 drastically diminished the cytoplasmic localization of RecQL4. Strikingly, addition of ubiquitination tail at the C-terminus of RecQL4 substantially enriched the cytoplasmic fraction of RecQL4 only in the presence of functional pNES2. Immunofluorescence studies revealed that the cytoplasmic RecQL4 was localized in mitochondria. Consistent with its mitochondrial localization, a regulatory role for RecQL4 in the maintenance of mitochondrial DNA (mtDNA) copy number was demonstrated. Elevation of ectopic expression of RecQL4 increased the mtDNA copy number in HEK293 cells while RecQL4 knock down markedly decreased the mtDNA copy number in U2OS cells. Additionally, a substantially increased level of mitochondrial superoxide production, and a markedly decreased repair capacity for oxidative DNA damage were observed in the mitochondria of both RecQL4 deficient human fibroblasts and RecQL4-suppressed cancer cells. These data strongly suggest a regulatory role for RecQL4 in mitochondrial stability and function. Collectively, our study demonstrates that NES-mediated RecQL4 export to the cytoplasm is essential for the maintenance of mitochondrial genome stability.
The international journal of biochemistry & cell biology 07/2012; 44(11):1942-51. · 4.89 Impact Factor
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Cui Yang,
Aili Li, Yongliang Zhao,
Zenglin Zhang,
Yuanfang Zhu,
Xiaomei Tan,
Shuaifeng Geng,
Hanzi Guo,
Xueyong Zhang,
Zhensheng Kang,
Long Mao
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[hide abstract]
ABSTRACT: Abscisic acid (ABA) plays pivotal roles in plant biotic and abiotic stress responses, where calcium ions are important second
messengers. Calcium/calmodulin-dependent protein kinase (CCaMK) is essential for nodulation in legumes, but whether it will
perceive calcium signals from abiotic stresses is not clear, especially in non-legume plants. Here we report the isolation
and characterization of the D-genome copy of wheat CCaMK gene TaCCaMK. TaCCaMK was predominantly expressed in root tissues of wheat seedlings, and its proteins were located both on the cytoplasm membrane
and in the nucleus as shown in the onion epidermis cells. Quantitative real-time reverse transcriptase polymerase chain reaction
(qRT-PCR) assay showed that the expression of TaCCaMK was downregulated by ABA, as well as NaCl and PEG treatments in wheat seedling roots. A DNA fragment of 1,119bp upstream
of the start codon of the TaCCaMK gene (pTaCCaMK) was isolated by thermal asymmetric interlaced PCR (TAIL-PCR), on which six ABA-responsive cis-elements were predicted. pTaCCaMK can drive GUS reporter gene expression in the root and stem stalk of the transgenic Arabidopsis plants which can be repressed by ABA treatments, consistent with the observation in the qRT-PCR assay in wheat. Overexpressing
TaCCaMK in Arabidopsis plants reduced their sensitivity to ABA treatment during seed germination and root elongation. Under high-salt conditions,
the transgenic plants also conferred enhanced seed germination rate and became hypersensitive with increased chlorosis. Therefore,
our data suggest that TaCCaMK is a negative regulator for ABA signaling which may participate in abiotic stress responses in wheat.
KeywordsWheat–Calcium–
CCaMK
–ABA–Abiotic stress
Plant Molecular Biology Reporter 04/2012; 29(3):681-692. · 2.45 Impact Factor
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ABSTRACT: Gene duplication contributes to the expansion of gene families and subsequent functional diversification. Calcium-dependent protein kinases (CDPKs) are members of an important calcium sensor family involved in abiotic and biotic stress signaling in plants. We report here the molecular evolution and expression analysis of a pair of duplicated CDPK genes CPK7 and CPK12 that arose in the common ancestor of grass species. With higher nonsynonymous/synonymous ratios (dN/dS, or ω), CPK12 genes appear to diverge more rapidly than CPK7s, suggesting relaxed selection constraints on CPK12s. Sliding window analysis revealed increased dN and ω values at N-terminal regions and the calcium-binding EF hand loops. Likelihood analyses using various models in PAML 4.0 showed purifying selection on both CPK7 and CPK12 lineages. In addition to the divergence in cis-element combinations on their promoters, functional divergence of CPK7 and CPK12 genes was also observed in wheat where TaCPK7 was found to respond to drought (PEG), salt (NaCl), cold, and hydrogen peroxide (H(2)O(2)) while TaCPK12 responded only to the treatment of ABA, a feature that may complement or expand TaCPK7-mediated stress signaling networks of wheat. The contrasting expression patterns of CPK7 and CPK12 genes under stress conditions were also observed in rice, suggesting conservative functional evolution of these genes. Since no positive selection was detected between the two lineages, the divergence of CPK7 and CPK12 genes should be ascribed to subfunctionalization, rather than neofunctionalization. Thus, our work demonstrates another case of evolutionary employment of duplicated genes via subfunctionalization for better adaptation.
Gene 01/2011; 475(2):94-103. · 2.34 Impact Factor
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[show abstract]
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ABSTRACT: The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 (α-ACTNl) expression in human T cells. α-ACTNl RNAi resulted in inhibited chemotaxis to SDF-lα, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTNl (rr-α-ACTNl) in the same cell. The presence of a GFP tag on rr-α-ACTNl allowed for detection of appropriate subcellular localization of rr-α-ACTNl. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.
Genomics Proteomics & Bioinformatics 12/2010; 8(4):238-45.
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[show abstract]
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ABSTRACT: The direct negative impact of the transcriptional activity of one component on the second one in cis is referred to as transcriptional interference (TI). U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged U6 promoter driving shRNA expression and UbiC promoter in two promoter arrangements. In primary macrophages, we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing U6 promoter activity, CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter. Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrangement-dependent manner.
Genomics Proteomics & Bioinformatics 09/2010; 8(3):170-9.
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Cui Yang,
Aili Li, Yongliang Zhao,
Zenglin Zhang,
Yuanfang Zhu,
Xiaomei,
Shuaifeng Geng,
Hanzi Guo,
Xueyong Zhang,
Zhensheng Kang,
Long Mao
[show abstract]
[hide abstract]
ABSTRACT: Abscisic acid (ABA) plays pivotal roles in plant biotic and abiotic stress responses, where calcium ions are important second messengers. Calcium/calmodulin-dependent protein kinase (CCaMK) is essential for nodulation in legumes, but whether it will perceive calcium signals from abiotic stresses is not clear, especially in non-legume plants. Here we report the isolation and characterization of the D-genome copy of wheat CCaMK gene TaCCaMK. TaCCaMK was predom-inantly expressed in root tissues of wheat seedlings, and its proteins were located both on the cytoplasm membrane and in the nucleus as shown in the onion epidermis cells. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay showed that the expression of TaCCaMK was downregulated by ABA, as well as NaCl and PEG treatments in wheat seedling roots. A DNA fragment of 1,119 bp upstream of the start codon of the TaCCaMK gene (pTaCCaMK) was isolated by thermal asymmetric interlaced PCR (TAIL-PCR), on which six ABA-responsive cis-elements were predicted. pTaCCaMK can drive GUS reporter gene expression in the root and stem stalk of the transgenic Arabidopsis plants which can be repressed by ABA treatments, consistent with the observation in the qRT-PCR assay in wheat. Overexpress-ing TaCCaMK in Arabidopsis plants reduced their sensi-tivity to ABA treatment during seed germination and root elongation. Under high-salt conditions, the transgenic plants also conferred enhanced seed germination rate and became hypersensitive with increased chlorosis. There-fore, our data suggest that TaCCaMK is a negative regulator for ABA signaling which may participate in abiotic stress responses in wheat.
