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Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

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      Abstract

      Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

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      Most cited references 32

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      GATEWAY vectors for Agrobacterium-mediated plant transformation.

      Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.
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        Introns and the origin of nucleus-cytosol compartmentalization.

        The origin of the eukaryotic nucleus marked a seminal evolutionary transition. We propose that the nuclear envelope's incipient function was to allow mRNA splicing, which is slow, to go to completion so that translation, which is fast, would occur only on mRNA with intact reading frames. The rapid, fortuitous spread of introns following the origin of mitochondria is adduced as the selective pressure that forged nucleus-cytosol compartmentalization.
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          A knowledge base for predicting protein localization sites in eukaryotic cells.

          To automate examination of massive amounts of sequence data for biological function, it is important to computerize interpretation based on empirical knowledge of sequence-function relationships. For this purpose, we have been constructing a knowledge base by organizing various experimental and computational observations as a collection of if-then rules. Here we report an expert system, which utilizes this knowledge base, for predicting localization sites of proteins only from the information on the amino acid sequence and the source origin. We collected data for 401 eukaryotic proteins with known localization sites (subcellular and extracellular) and divided them into training data and testing data. Fourteen localization sites were distinguished for animal cells and 17 for plant cells. When sorting signals were not well characterized experimentally, various sequence features were computationally derived from the training data. It was found that 66% of the training data and 59% of the testing data were correctly predicted by our expert system. This artificial intelligence approach is powerful and flexible enough to be used in genome analyses.
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            Author and article information

            Affiliations
            [1 ]Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín Grupo de Ecología Genética, Granada, Spain
            [2 ]Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Gif-sur-Yvette, France
            University of Toronto, Canada
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: RNM JIJZ MC NT. Performed the experiments: RNM PL JIJZ. Analyzed the data: RNM MC FF NT. Contributed reagents/materials/analysis tools: MC FF NT. Wrote the paper: RNM JIJZ NT.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2013
            31 December 2013
            : 8
            : 12
            3877140
            PONE-D-13-42132
            10.1371/journal.pone.0084056
            (Editor)

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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            Pages: 8
            Funding
            This work was supported by research grants CSD 2009-0006 from the Consolider-Ingenio program, including ERDF (European Regional Development Funds), BIO2011-24401 from the, currently Ministerio de Economía y Competitividad and CSIC-CNRS bilateral projects 2002FR0032 and 2005FR0004. RNM held an FPI fellowship from the Spanish Ministerio de Ciencia e Innovación. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology
            Biotechnology
            Applied Microbiology
            Genetic Engineering
            Computational Biology
            Molecular Genetics
            Evolutionary Biology
            Genetics
            Molecular Genetics
            Microbiology
            Bacteriology
            Bacterial Biochemistry
            Model Organisms
            Plant and Algal Models
            Arabidopsis Thaliana
            Molecular Cell Biology

            Uncategorized

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