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RNA-binding Protein (RNA-bind + protein)
Selected AbstractsCorrelation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouseDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2005Rieko Asai Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium. [source] Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacyFEBS JOURNAL, Issue 22 2009Hemant K. Kini To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5, end is less stably hybridized relative to the end containing the passenger strand 5, end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5, end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5, end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5, end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique. [source] Xenopus Rbm9 is a novel interactor of XGld2 in the cytoplasmic polyadenylation complexFEBS JOURNAL, Issue 3 2008Catherine Papin During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes. [source] A trans -acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigeneFEBS JOURNAL, Issue 13 2000Andrej Poleev Two clones were isolated in a three-hybrid screen of a rat fetal brain P5 cDNA library with an intronic splicing enhancer of the amyloid precursor protein (APP) gene as RNA bait. These clones represent the rat homologues of the previously described genes CUG-binding protein (CUG-BP) and Siah-binding protein (Siah-BP). Both interact in a sequence-specific manner with the RNA bait used for library screening as well as with the CUG repeat. In contrast, no interactions were observed in the three-hybrid assay with other baits tested. In two-hybrid assays, Siah-BP interacts with U2AF65 as well as with itself. EWS, an RGG-type RNA-binding protein associated with Ewing sarcoma, was identified as an interacting partner for the CUG-BP homologue in a two-hybrid assay for protein,protein interactions performed with various factors involved in RNA metabolism. Splicing assays performed by RT-PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a reporter, indicate exclusion of exon 8 if the CUG-BP homologue is present. We conclude that clone AF169013 and its counterpart in human CUG-BP could be the trans -acting factors that interact with the splicing enhancer downstream of exon 8, and in this way influence alternative splicing of the APP minigene. [source] Retrieval of first genome data for rice cluster I methanogens by a combination of cultivation and molecular techniquesFEMS MICROBIOLOGY ECOLOGY, Issue 2 2005Christoph Erkel Abstract We report first insights into a representative genome of rice cluster I (RC-I), a major group of as-yet uncultured methanogens. The starting point of our study was the methanogenic consortium MRE50 that had been stably maintained for 3 years by consecutive transfers to fresh medium and anaerobic incubation at 50 °C. Process-oriented measurements provided evidence for hydrogenotrophic CO2 -reducing methanogenesis. Assessment of the diversity of consortium MRE50 suggested members of the families Thermoanaerobacteriaceae and Clostridiaceae to constitute the major bacterial component, while the archaeal population was represented entirely by RC-I. The RC-I population amounted to more than 50% of total cells, as concluded from fluorescence in situ hybridization using specific probes for either Bacteria or Archaea. The high enrichment status of RC-I prompted construction of a large insert fosmid library from consortium MRE50. Comparative sequence analysis of internal transcribed spacer (ITS) regions revealed that three different RC-I rrn operon variants were present in the fosmid library. Three, approximately 40-kb genomic fragments, each representative for one of the three different rrn operon variants, were recovered and sequenced. Computational analysis of the sequence data resulted in two major findings: (i) consortium MRE50 most likely harbours only a single RC-I genotype, which is characterized by multiple rrn operon copies; (ii) seven genes were identified to possess a strong phylogenetic signal (eIF2a, dnaG, priA, pcrA, gatD, gatE, and a gene encoding a putative RNA-binding protein). Trees exemplarily computed for the deduced amino acid sequences of eIF2a, dnaG, and priA corroborated a specific phylogenetic association of RC-I with the Methanosarcinales. [source] hnRNP K interacts with RNA binding motif protein 42 and functions in the maintenance of cellular ATP level during stress conditionsGENES TO CELLS, Issue 2 2009Toshiyuki Fukuda Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a conserved RNA-binding protein that is involved in multiple processes of gene expression, including chromatin remodeling, transcription, RNA splicing, mRNA stability and translation, together with diverse groups of molecular partners. Here we identified a previously uncharacterized protein RNA binding motif protein 42 (RBM42) as hnRNP K-binding protein. RBM42 directly bound to hnRNP K in vivo and in vitro. RBM42 also directly bound to the 3, untranslated region of p21 mRNA, one of the target mRNAs for hnRNP K. RBM42 predominantly localized within the nucleus and co-localized with hnRNP K there. When cells were treated with agents, puromycin, sorbitol or arsenite, which induced the formation of stress granules (SGs), cytoplasmic aggregates of stalled translational pre-initiation complexes, both hnRNP K and RBM42 localized at SGs. Depletion of hnRNP K by RNA interference decreased cellular ATP level following release from stress conditions. Simultaneous depletion of RBM42 with hnRNP K enhanced the effect of the hnRNP K depletion. Our results indicate that hnRNP K and RBM42 are components of SGs and suggest that hnRNP K and RBM42 have a role in the maintenance of cellular ATP level in the stress conditions possibly through protecting their target mRNAs. [source] MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegansGENES TO CELLS, Issue 11 2000Akinori Yoda Neural RNA-binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post-transcriptional gene regulation. RNA-binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood. We identified a Caenorhabditis elegans Musashi homologue, MSI-1, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi-1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurones required for turning and vulva location. However, msi-1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. Our results suggest that msi-1 is expressed specifically in postmitotic neurones in C. elegans. msi-1 is required for full development of male mating behaviour, possibly through regulation of msi-1 expressing neurones. [source] FMRP RNA targets: identification and validationGENES, BRAIN AND BEHAVIOR, Issue 6 2005J. C. Darnell The Fragile X Syndrome is caused by the loss of function of the FMR1 gene (Pieretti et al. 1991. Cell 66, 817,822; O'Donnell & Warren 2002. Annu Rev Neurosci 25, 315,338]. Identification of the RNA targets to which FMRP binds is a key step in understanding the function of the protein and the cellular defects caused by its absence (Darnell et al. 2004 Ment Retard Dev Disabil Res Rev 10, 49,52). Here we discuss the current understanding of FMRP as an RNA-binding protein, the different approaches that have been taken to identify FMRP RNA targets and the relevance of some of these approaches to FMRP biology. In addition, we present evidence that point mutations in the K-homology (KH)1 or KH2 domains of FMRP abrogate its polyribosome association in transfected neuroblastoma cells but that the deletion of the RGG box does not. This suggests that RNA binding by the RGG box of FMRP may mediate other aspects of cellular mRNA metabolism such as mRNA localization or that it may have a role downstream of polyribosome association. [source] Expression of the RNA-binding protein Musashi1 in adult liver stem-like cellsHEPATOLOGY RESEARCH, Issue 4 2010Etsuko Hattori Aim:, Musashi1 is an RNA-binding protein that regulates the Notch signaling pathway in stem cells. Our previous study revealed that Musashi1 is expressed in early hepatocytes during liver development in the mouse. However, whether this unique protein is expressed with Notch signaling markers in adult liver stem-like cells remains unknown. Methods:, Established hepatic stem-like cells (HSLC), which were derived from adult Sprague,Dawley rats, were used for experiments in vitro. HSLC were differentiated into mature cells in terms of producing albumin when co-cultured with epidermal growth factor (EGF). The mRNA expression of Musashi1, Notch family (Notch1 and Notch2), Jagged1 and Hes1 was examined in HSLC before and after cell differentiation using polymerase chain reaction-based techniques. Protein expression of Musashi1 was examined in the HSLC and normal mature hepatocytes by immunofluorescence staining. Results:, The mRNA expression of Musashi1, Notch1, Jagged1 and Hes1 was detected in the original HSLC before culturing with EGF but not in primary cultured mature hepatocytes. The mRNA expression of Musashi1 and Hes1 was found to be downregulated in differentiated HSLC that produce albumin. Protein expression of Musashi1 was detectable in the original HSLC but not in both differentiated HSLC and mature hepatocytes. Conclusion:, These findings demonstrate that the RNA-binding protein Musashi1 is expressed with Notch signaling markers in adult liver stem-like cells. [source] MSY2 and polypyrimidine tract binding protein 2 stabilize mRNAs in the mammalian testisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2008Mingang Xu Summary MSY2 is a highly conserved and abundant DNA/RNA-binding protein that functions as a global stabilizer/translational suppressor of mRNAs in male germ cells. The polypyrimidine tract binding protein, PTBP2, is an RNA-binding protein that splices nuclear transcripts and stabilizes specific mRNAs in the cytoplasm. The mechanisms whereby MSY2 selects and stabilizes a large group of male germ cell mRNAs and PTBP2 stabilizes specific mRNAs such as the phosphoglycerate kinase 2 mRNA in the testis and in transfected cells will be discussed. [source] 2,,3,-Cyclic nucleotide 3,-phosphodiesterase: A novel RNA-binding protein that inhibits protein synthesisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2009Michel Gravel Abstract 2,,3,-Cyclic nucleotide 3,-phosphodiesterase (CNP) is one of the earliest myelin-related proteins to be specifically expressed in differentiating oligodendrocytes (ODCs) in the central nervous system (CNS) and is implicated in myelin biogenesis. CNP possesses an in vitro enzymatic activity, whose in vivo relevance remains to be defined, because substrates with 2,,3,-cyclic termini have not yet been identified. To characterize CNP function better, we previously determined the structure of the CNP catalytic domain by NMR. Interestingly, the structure is remarkably similar to the plant cyclic nucleotide phosphodiesterase (CPDase) from A. thaliana and the bacterial 2,-5, RNA ligase from T. thermophilus, which are known to play roles in RNA metabolism. Here we show that CNP is an RNA-binding protein. Furthermore, by using precipitation analyses, we demonstrate that CNP associates with poly(A)+ mRNAs in vivo and suppresses translation in vitro in a dose-dependent manner. With SELEX, we isolated RNA aptamers that can suppress the inhibitory effect of CNP on translation. We also demonstrate that CNP1 can bridge an association between tubulin and RNA. These results suggest that CNP1 may regulate expression of mRNAs in ODCs of the CNS. © 2008 Wiley-Liss, Inc. [source] Molecular phenotype of Fragile X syndrome: FMRP, FXRPs, and protein targetsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Walter E. Kaufmann Abstract Fragile X syndrome (FraX) is one of the most prevalent genetic causes of mental retardation. FraX is associated with an unstable expansion of a polymorphism within the 5, untranslated region of the FMR1 gene. The main consequence of this mutation is a reduction in the levels of the gene product (FMRP). FMRP is an RNA-binding protein with multiple spliced variants (isoforms) and high levels of expression in a variety of tissues, including neurons. In the latter cells, it is localized not only to the perikaryon but also to dendrites and dendritic spines. FMRP belongs to a family of proteins that includes the Fragile X Related Proteins or FXRPs. FXRPs share high homology in their functional domains with FMRP, and also associate with mRNA and components of the protein synthesis apparatus. However, FXRPs do not have the same temporo-spatial pattern of distribution (and other properties) of FMRP. Immunochemical assays have confirmed that a functionally uncompensated FMRP deficit is the essence of the FraX molecular phenotype. Here, we report our preliminary study on FXRPs levels in leukocytes from FraX males. By immunoblotting, we found that a marked reduction in FMRP levels is associated with a modest increase in FXR1P and no changes in FXR2P levels. The consequences of this reduced FMRP expression on protein synthesis, in other words, the identification of FMRP targets, can be studied by different molecular approaches including protein interaction and proteomics methods. By two-dimensional gel electrophoresis, we showed that in FraX leukocytes there is a defect in acetylation that involves prominently the regulatory protein annexin-1. Extension of current studies of the molecular phenotype to more brain-relevant tissue samples, a wider range of proteomics-based methods, and correlative analyses of FMRP homologues and FMRP targets with multiple behavioral measures, will greatly expand our understanding of FraX pathogenesis and it will help to develop and monitor new therapeutic strategies. Microsc. Res. Tech. 57:135,144, 2002. © 2002 Wiley-Liss, Inc. [source] Fragile X mental retardation: Misregulation of protein synthesis in the developing brain?MICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Yue FengArticle first published online: 18 APR 200 Abstract Fragile X mental retardation results from the absence of a selective RNA-binding protein, FMRP. Previous studies demonstrated that FMRP forms messenger ribonucleoprotein (mRNP) complexes to associate with translating polyribosomes, suggesting that FMRP is involved in regulating protein synthesis. We are now facing the changing questions: How does FMRP influence protein synthesis in the brain? What is the target for FMRP in learning and memory? How does the absence of FMRP cause misregulation of protein synthesis, which in turn leads to mental impairment in fragile X syndrome? Models for abnormal neuronal function as a result of misregulated translation due to the absence of FMRP are discussed. Microsc. Res. Tech. 57:145,147, 2002. © 2002 Wiley-Liss, Inc. [source] Mice deficient for RNA-binding protein brunol1 show reduction of spermatogenesis but are fertileMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 11 2007Arvind Dev Abstract RNA-binding proteins are involved in post-transcriptional processes like mRNA stabilization, alternative splicing, and transport. Brunol1 is a novel mouse gene related to elav/Bruno family of genes encoding for RNA-binding proteins. We report here the expression and functional analysis of murine Brunol1. Expression analysis of Brunol1 during embryogenesis by RT-PCR showed that Brunol1 expression starts at 9.5 dpc and continues to the later stages of embryonic development. In adult mice, the Brunol1 expression is restricted to brain and testis. We also analyzed the Brunol1 expression in testes of different mutants with spermatogenesis defects: W/WV, Tfm/y, Leyl,/,, olt/olt, and qk/qk. Brunol1 transcript was detectable in Leyl,/,, olt/olt, and qk/qk mutant but not in W/WV and Tfm/y mutants. We also showed by transfection of a fusion protein of green fluorescent protein and Brunol1 protein into NIH3T3 cells, that Brunol1 is localized in cytoplasm and nucleus. In order to elucidate the function of the Brunol1 protein in spermatogenesis, we disrupted the Brunol1 locus in mouse by homologous recombination, which resulted in a complete loss of the Brunol1 transcript. Male and female Brunol1+/, and Brunol1,/, mice from genetic backgrounds C57BL/6J,×,129/Sv hybrid and 129X1/SvJ when inbred exhibited normal phenotype and are fertile, although the number and motility of sperms are significantly reduced. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. Collectively our results demonstrate that Brunol1 might be nonessential protein for mouse embryonic development and spermatogenesis. Mol. Reprod. Dev. 74: 1456,1464, 2007. © 2007 Wiley-Liss, Inc. [source] Solution structure of the region 51,160 of human KIN17 reveals an atypical winged helix domainPROTEIN SCIENCE, Issue 12 2007Ludovic Carlier Abstract Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28,50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268,393) only found in higher eukaryotes. Here we report the solution structure of the region 51,160 of human KIN17. We show that this fragment folds into a three-,-helix bundle packed against a three-stranded ,-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 310 -helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51,160 might rather be involved in protein,protein interaction through its conserved surface centered on the 310 -helix. [source] Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defensePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Ivy Widjaja Abstract Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone-responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2-DE facilitated the discovery of low abundance proteins , enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA-binding protein, and a C2-domain-containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA-binding protein, involvement of PTM and post-transcriptional regulation are implicated, respectively. [source] Identification of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatulaTHE PLANT JOURNAL, Issue 1 2007Francisco Merchan Summary Root growth and function are determined by the action of environmental stresses through specific genes that adapt root development to these restrictive conditions. We have defined in vitro conditions affecting the growth and recovery of Medicago truncatula roots after a salt stress. A dedicated macroarray containing 384 genes, based on a large-scale subtractive hybridization approach, was constructed and used to analyze gene expression during salt stress and recovery of root growth from this stress. Several potential regulatory genes were identified as being linked to this recovery process: a novel RNA-binding protein, a small G-protein homologous to ROP9, a receptor-like kinase, two TF IIIA-like and an AP2-like transcription factors (TF), MtZpt2-1, MtZpt2-2 and MtAp2, and a histidine kinase associated with cytokinin transduction pathways. The two ZPT2-type TFs were also rapidly induced by cold stress in roots. By analyzing transgenic M. truncatula plants showing reduced expression levels of both TFs and affected in their capacity to recover root growth after a salt stress, we identified potential target genes that were either activated or repressed in these plants. Overexpression of MtZpt2-1 in roots conferred salt tolerance and affected the expression of three putative targets in the predicted manner: a cold-regulated A (CORA) homolog, a flower-promoting factor (FPF1) homolog and an auxin-induced proline-rich protein (PRP) gene. Hence, regulatory networks depending on TFIIIA-like transcription factors are involved in the control of root adaptation to salt stress. [source] SERRATE is a novel nuclear regulator in primary microRNA processing in ArabidopsisTHE PLANT JOURNAL, Issue 6 2006Li Yang Summary The Arabidopsis gene SERRATE (SE) controls leaf development, meristem activity, inflorescence architecture and developmental phase transition. It has been suggested that SE, which encodes a C2H2 zinc finger protein, may change gene expression via chromatin modification. Recently, SE has also been shown to regulate specific microRNAs (miRNAs), miR165/166, and thus control shoot meristem function and leaf polarity. However, it remains unclear whether and how SE modulates specific miRNA processing. Here we show that the se mutant exhibits some similar developmental abnormalities as the hyponastic leaves1 (hyl1) mutant. Since HYL1 is a nuclear double-stranded RNA-binding protein acting in the DICER-LIKE1 (DCL1) complex to regulate the first step of primary miRNA transcript (pri-miRNA) processing, we hypothesized that SE could play a previously unrecognized and general role in miRNA processing. Genetic analysis supports that SE and HYL1 act in the same pathway to regulate plant development. Consistently, SE is critical for the accumulation of multiple miRNAs and the trans -acting small interfering RNA (ta-siRNA), but is not required for sense post-transcriptional gene silencing. We further demonstrate that SE is localized in the nucleus and interacts physically with HYL1. Finally, we provide evidence that SE and HYL1 probably act with DCL1 in processing pri-miRNAs before HEN1 in miRNA biogenesis. In plants and animals, miRNAs are known to be processed in a stepwise manner from pri-miRNA. Our data strongly suggest that SE plays an important and general role in pri-miRNA processing, and it would be interesting to determine whether animal SE homologues may play similar roles in vivo. [source] Cold-inducible zinc finger-containing glycine-rich RNA-binding protein contributes to the enhancement of freezing tolerance in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 6 2005Yeon-Ok Kim Summary Glycine-rich RNA-binding proteins (GR-RBPs) have been implicated to play roles in post-transcriptional regulation of gene expression in plants under various stress conditions, but the functional roles of GR-RBPs under stress conditions remain to be verified. Here, we examine the biological roles of a GR-RBP, designated atRZ-1a, in Arabidopsis thaliana under stress conditions. atRZ-1a was expressed ubiquitously in various Arabidopsis organs including stems, roots, leaves, flowers, and siliques. The transcript level of atRZ-1a increased markedly by cold stress, whereas its expression was marginally downregulated by drought stress or abscisic acid treatment. Germination and seedling growth of the loss-of-function mutants were retarded remarkably compared with those of the wild type under cold stress. In contrast, the transgenic Arabidopsis plants that overexpress atRZ-1a displayed earlier germination and better seedling growth than the wild type under cold stress. Moreover, the atRZ-1a-overexpressing transgenic Arabidopsis plants were more freezing tolerant than the wild-type plants. Heterologous expression of atRZ-1a in Escherichia coli demonstrated that the E. coli cells expressing atRZ-1a displayed much higher growth rate than the non-transformed cells after cold shock. These results provide evidence that atRZ-1a affects seed germination and seedling growth under low temperature and plays a role in the enhancement of freezing tolerance in Arabidopsis plants. [source] Mouse testis,brain RNA-binding protein (TB-RBP): expression, purification and crystal X-ray diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001John M. Pascal TB-RBP (testis,brain RNA-binding protein) is a mouse RNA-binding protein that controls the temporal and spatial expression of mRNA. Found most abundantly in brain and male germ cells in the testis, TB-RBP is known to suppress the translation of specific testicular and brain mRNAs as part of cell development. TB-RBP,mRNA complexes can bind microtubules and thereby facilitate RNA translocation. Translin is the human orthologue of TB-RBP which binds to single-stranded ends of DNA sequences in breakpoint regions of chromosomal translocations. TB-RBP/translin has been crystallized in space group P21212. The expression, purification, and crystallization of TB-RBP are described as well as preliminary X-ray diffraction data. The multimeric state of TB-RBP is addressed using dynamic light-scattering results. [source] Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Seiki Baba The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq,RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq,RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13,Å, while the type 2 Hfq,RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92,Å. Diffraction data were collected to a resolution of 2.20,Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis. [source] mRNPs take shape by CLIPPING and PAIRINGBIOESSAYS, Issue 11 2006Robert B. Denman The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations. BioEssays 28: 1132,1143, 2006. © 2006 Wiley Periodicals, Inc. [source] Purification, crystallization and initial X-ray diffraction study of the zinc-finger domain of zebrafish NanosACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Hiroshi Hashimoto Nanos is a highly conserved RNA-binding protein in higher eukaryotes and acts as a key regulator protein involved in translational control utilizing the 3, untranslated region of mRNA. The C-terminal domain of Nanos has two conserved and novel CCHC-type zinc-finger motifs that are responsible for the function of Nanos. To clarify the structural basis of the function of Nanos, the C-terminal domain (residues 59,159) of zebrafish Nanos was overexpressed, purified and crystallized. The crystal belonged to space group P63, with unit-cell parameters a = b = 100.9, c = 71.5,Å, , = 120°. Structure determination by the MAD/SAD method is now in progress. [source] Crystallization and preliminary crystallographic analysis of the second RRM of Pub1 from Saccharomyces cerevisiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Yingji Cui mRNA stability is elaborately regulated by elements in the mRNA transcripts and their cognate RNA-binding proteins, which play important roles in regulating gene expression at the post-transcriptional level in eukaryotes. Poly(U)-binding protein 1 (Pub1), which is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae, is involved in the regulation of mRNA turnover as a trans -acting factor. It binds to transcripts containing the AU-rich element in order to protect them from degradation. Pub1 contains three RNA-recognition motifs (RRMs) which play significant roles in mRNA binding at AU-rich elements and stabilizer elements. In this study, the second RRM of Pub1 was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283,K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group H3. [source] Anthrax lethal toxin promotes dephosphorylation of TTP and formation of processing bodiesCELLULAR MICROBIOLOGY, Issue 4 2010Edith M. C. Chow Summary Anthrax lethal toxin (LeTx) is composed of protective antigen (PA) and lethal factor (LF) , PA is the receptor-binding moiety and LF is a protease that cleaves mitogen-activated protein kinase kinases (MAPKKs). LeTx subverts the immune response to Bacillus anthracis in several ways, such as downregulating interleukin-8 (IL-8) by increasing the rate of IL-8 mRNA degradation. Many transcripts are regulated through cis -acting elements that bind proteins that either impede or promote degradation. Some of these RNA-binding proteins are regulated by MAPKs and previous work has demonstrated that interfering with MAPK signalling decreases the half-life of IL-8 mRNA. Here, we have localized a segment within the IL-8 3, untranslated region responsible for LeTx-induced transcript destabilization and show that this is caused by inhibition of the p38, ERK and JNK pathways. TTP, an RNA-binding protein involved in IL-8 mRNA decay, became hypophosphorylated in LeTx-treated cells and knock-down of TTP prevented LeTx from destabilizing the IL-8 transcript. Cells that were treated with LeTx exhibited increased localization of TTP to Processing bodies, which are structures that accumulate transcripts targeted for degradation. We furthermore observed that LeTx promoted the formation of Processing bodies, revealing a link between the toxin and a major mRNA decay pathway. [source] Cellular oxygen sensing, signalling and how to survive translational arrest in hypoxiaACTA PHYSIOLOGICA, Issue 2 2009M. Fähling Abstract Hypoxia is a consequence of inadequate oxygen availability. At the cellular level, lowered oxygen concentration activates signal cascades including numerous receptors, ion channels, second messengers, as well as several protein kinases and phosphatases. This, in turn, activates trans -factors like transcription factors, RNA-binding proteins and miRNAs, mediating an alteration in gene expression control. Each cell type has its unique constellation of oxygen sensors, couplers and effectors that determine the activation and predominance of several independent hypoxia-sensitive pathways. Hence, altered gene expression patterns in hypoxia result from a complex regulatory network with multiple divergences and convergences. Although hundreds of genes are activated by transcriptional control in hypoxia, metabolic rate depression, as a consequence of reduced ATP level, causes inhibition of mRNA translation. In a multi-phase response to hypoxia, global protein synthesis is suppressed, mainly by phosphorylation of eIF2-alpha by PERK and inhibition of mTOR, causing suppression of 5,-cap-dependent mRNA translation. Growing evidence suggests that mRNAs undergo sorting at stress granules, which determines the fate of mRNA as to whether being translated, stored, or degraded. Data indicate that translation is suppressed only at ,free' polysomes, but is active at subsets of membrane-bound ribosomes. The recruitment of specific mRNAs into subcellular compartments seems to be crucial for local mRNA translation in prolonged hypoxia. Furthermore, ribosomes themselves may play a significant role in targeting mRNAs for translation. This review summarizes the multiple facets of the cellular adaptation to hypoxia observed in mammals. [source] Two splicing isoforms of the Y-box protein ctYB-1 appear on the same mRNA moleculeFEBS JOURNAL, Issue 1 2007Dmitry Nashchekin Y-box proteins constitute an evolutionarily conserved family of DNA- and RNA-binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the vertebrate Y-box protein YB-1 was recently characterized and designated ctYB-1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We further analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT-PCR analysis suggested that there is just one YB-1 gene, and that the two size variants represent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth-instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35,40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50-specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional significance of the two isoforms is being discussed. [source] MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegansGENES TO CELLS, Issue 11 2000Akinori Yoda Neural RNA-binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post-transcriptional gene regulation. RNA-binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood. We identified a Caenorhabditis elegans Musashi homologue, MSI-1, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi-1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurones required for turning and vulva location. However, msi-1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. Our results suggest that msi-1 is expressed specifically in postmitotic neurones in C. elegans. msi-1 is required for full development of male mating behaviour, possibly through regulation of msi-1 expressing neurones. [source] A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian populationINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2006K. Thangaraj Summary The autosomal DAZL (Deleted-in- Azoospermic- Like) gene, mapped to the short arm of the human chromosome 3, is the precursor for the Y-chromosomal DAZ cluster, which encodes for putative RNA-binding proteins. Mutations in the DAZL have been reported to be associated with spermatogenic failure in Taiwanese population but not in Caucasians. As there was no study on Indian populations, we have analysed the entire coding sequences of exons 2 and 3 of DAZL in a total of 1010 men from Indian subcontinent, including 660 infertile men with 598 non-obstructive azoospermia, 62 severe oligozoospermia and 350 normozoospermic fertile control men, to investigate whether mutation(s) in the DAZL is associated with male infertility. Interestingly, none of our samples (1010) showed A386G (T54A) mutation, which was found to be associated with spermatogenic failure in Taiwanese population. In contrast, A260G (T12A) mutation was observed in both infertile and normozoospermic fertile control men, without any significant association with infertile groups (,2 = 0.342; p = 0.556). Similarly, we have found a novel A437G (I71V) mutation, which is also present in both infertile and normozoospermic fertile control men without any significant difference (,2 = 0.476; p = 0.490). Our study clearly demonstrates the complete absence of the A386G (T54A) mutation in Indian subcontinent and the other two mutations , A260G (T12A) and A437G (I71V) , observed are polymorpic. Therefore, we conclude that these mutations in the DAZL gene are not associated with male infertility in Indian subcontinent. [source] T-armless tRNAs and elongated elongation factor TuIUBMB LIFE, Issue 2 2007Takashi Ohtsuki Abstract Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins. IUBMB Life, 59: 68-75, 2007 [source] | ||||||||||||||||||||||||||||