Home About us Contact
|
Home About us Contact | ||
|
|
||
RNA Metabolism (rna + metabolism)
Selected AbstractsThe effect of hfq on global gene expression and virulence in Neisseria gonorrhoeaeFEBS JOURNAL, Issue 19 2009Manuela Dietrich Hfq is an RNA chaperone that functions as a pleiotropic regulator for RNA metabolism in bacteria. In several pathogenic bacteria, Hfq contributes indirectly to virulence by binding to riboregulators that modulate the stability or translation efficiency of RNA transcripts. To characterize the role of Hfq in the pathogenicity of Neisseria gonorrhoeae, we generated an N. gonorrhoeae hfq mutant. Infectivity and global changes in gene expression caused by the hfq mutation in N. gonorrhoeae strain MS11 were analyzed. Transcriptional analysis using a custom-made N. gonorrhoeae microarray revealed that 369 ORFs were differentially regulated in the hfq mutant, MS11hfq, in comparison with the wild-type strain (202 were upregulated, and 167 were downregulated). The loss-of-function mutation in hfq led to pleiotropic phenotypic effects, including an altered bacterial growth rate and reduced adherence to epithelial cells. Twitching motility and microcolony formation were not affected. Hfq also appears to play a minor role in inducing the inflammatory response of infected human epithelial cells. Interleukin-8 production was slightly decreased, and activation of c-Jun N-terminal kinase, a mitogen-activated protein kinase, was reduced in MS11hfq- infected epithelial cells in comparison with wild type-infected cells. However, activation of nuclear factor kappa B, extracellular signal-regulated kinase 1/2 and p38 remained unchanged. The data presented suggest that Hfq plays an important role as a post-transcriptional regulator in N. gonorrhoeae strain MS11 but does not contribute significantly to its virulence in cell culture models. [source] Ki-1/57 interacts with PRMT1 and is a substrate for arginine methylationFEBS JOURNAL, Issue 17 2006Dario O. Passos The human 57 kDa Ki-1 antigen (Ki-1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki-1/57 interacts with chromo-helicase DNA-binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki-1/57 in a yeast two-hybrid system was the protein arginine-methyltransferase-1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two-hybrid system we were able to identify Ki-1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki-1/57 and its putative paralog CGI-55 have two conserved Gly/Arg-rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine-methylation by PRMT1. We observed that all Ki-1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki-1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine-2,,3,-dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki-1/57, whereas its paralog CGI-55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two-hybrid assay is an effective system for identifying novel PRMT arginine-methylation substrates and may be successfully applied to other members of the growing family of PRMTs. [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] The DEAD-box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB-mediated transcriptionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Musarat Ishaq Abstract DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-,B), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-,B dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-,B-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-,B-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-,B-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-,B transcriptional activity. J. Cell. Biochem. 106: 296,305, 2009. © 2008 Wiley-Liss, Inc. [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] An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2006Olivier Guipaud Abstract Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma,(EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor,(PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in,vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control. [source] Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platformPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2006Anja Tabbert Abstract During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N -nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein. [source] Post-translational modifications, but not transcriptional regulation, of major chloroplast RNA-binding proteins are related to Arabidopsis seedling developmentPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2006Bai-Chen Wang Abstract Chloroplast RNA-binding proteins are involved in stabilizing stored chloroplast mRNAs and in recruiting site-specific factors that mediate RNA metabolism. In the present study, we characterized two major chloroplast RNA-binding proteins, cp29A and cp29B, by MALDI-TOF MS, N-terminal sequencing, and ESI-MS/MS following 2D-PAGE separation. Polypeptides derived from cp29A were recovered with free N-terminus or with N-terminal acetylation. In addition to the two isoforms found for cp29A, an isoform derived from cp29B was also observed to have five amino acids cleaved from its N-terminus. Results of quantitative real-time RT-PCR indicate that both genes reached maximal rates of transcription 96,h after commencement of germination and maintained relatively high levels throughout the whole life cycle. Transcription of cp29A and cp29B did not vary significantly under light or dark conditions, although production of the acetylated and N-terminally cleaved protein isoforms exhibited light dependence. Exposure of etiolated Arabidopsis seedlings to light conditions for as short as 9,h restored the modified isoforms to levels similar to those found in green plants. Identification of post-translational modifications in major chloroplast RNA-binding proteins may help elucidate their roles in seedling development and in plant RNA stabilization during the greening process. [source] mRNA metabolism of flowering-time regulators in wild-type Arabidopsis revealed by a nuclear cap binding protein mutant, abh1THE PLANT JOURNAL, Issue 6 2007Josef M. Kuhn Summary The precise regulation of RNA metabolism has crucial roles in numerous developmental and physiological processes such as the induction of flowering in plants. Here we report the identification of processes associated with mRNA metabolism of flowering-time regulators in wild-type Arabidopsis plants, which were revealed by an early flowering mutation, abh1, in an Arabidopsis nuclear mRNA cap-binding protein. By using abh1 as an enhancer of mRNA metabolism events, we identify non-coding polyadenylated cis natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants. Our analyses also reveal a regulatory function of FLC intron 1 during transcript maturation in wild type. Moreover, transcripts encoding the FLM MADS box transcription factor are subject to premature intronic polyadenylation in wild type. In each case, abh1 showed altered patterns in RNA metabolism in these events compared with wild type. Together, abh1 enhances steps in the RNA metabolism that allowed us to identify novel molecular events of three key flowering-time regulators in wild-type plants, delivering important insights for further dissecting RNA-based mechanisms regulating flowering time in Arabidopsis. [source] Current hypotheses for the underlying biology of amyotrophic lateral sclerosis,ANNALS OF NEUROLOGY, Issue S1 2009Jeffrey D. Rothstein MD The mechanisms involved in selective motor neuron degeneration in amyotrophic lateral sclerosis remain unknown more than 135 years after the disease was first described. Although most cases have no known cause, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) have been implicated in a fraction of familial cases of the disease. Transgenic mouse models with mutations in the SOD1 gene and other ALS genes develop pathology reminiscent of the disorder, including progressive death of motor neurons, and have provided insight into the pathogenesis of the disease but have consistently failed to predict therapeutic efficacy in humans. However, emerging research has demonstrated that mutations and pathology associated with the TDP-43 gene and protein may be more common than SOD1 mutations in familial and sporadic ALS. Putative mechanisms of toxicity targeting motor neurons include oxidative damage, accumulation of intracellular aggregates, mitochondrial dysfunction, defects in axonal transport, growth factor deficiency, aberrant RNA metabolism, glial cell pathology, and glutamate excitotoxicity. Convergence of these pathways is likely to mediate disease onset and progression. Ann Neurol 2009;65 (suppl):S3,S9 [source] A birth-to-death view of mRNA from the RNA recognition motif perspective,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2008Terri Goss Kinzy Abstract RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process. [source] Mechanisms of RNA Degradation by the Eukaryotic ExosomeCHEMBIOCHEM, Issue 7 2010Rafal Tomecki Dr. RNA on its way to destruction: The exosome is a multi-subunit protein complex involved in essentially all phenomena associated with RNA metabolism in eukaryotic cells. This review discusses recent discoveries in the fields of biochemistry and structural biology that have shed new light on the mechanisms of RNA recruitment to the catalytic subunits of the exosome. [source] | |||||||||||||