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In vivo kinetics of U4/U6·U5 tri-snRNP formation in Cajal bodies

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      Abstract

      A combination of mathematical modeling and live-cell measurements was applied to determine the dynamics of small nuclear ribonucleoprotein (snRNP) formation in Cajal bodies of living cells. Our results indicate that a substantial fraction of tri-snRNPs is formed in Cajal bodies in cells with many Cajal bodies per nucleus.

      Abstract

      The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

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      Ribonucleoproteins (RNPs) mediate key cellular functions such as gene expression and its regulation. Whereas most RNP enzymes are stable in composition and harbor preformed active sites, the spliceosome, which removes noncoding introns from precursor messenger RNAs (pre-mRNAs), follows fundamentally different strategies. In order to provide both accuracy to the recognition of reactive splice sites in the pre-mRNA and flexibility to the choice of splice sites during alternative splicing, the spliceosome exhibits exceptional compositional and structural dynamics that are exploited during substrate-dependent complex assembly, catalytic activation, and active site remodeling.
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        The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.
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          Nuclear speckles: a model for nuclear organelles.

          Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA-protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.
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            Author and article information

            Affiliations
            aInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic
            bInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic
            cFaculty of Mathematics and Physics, Charles University, 121 16 Prague 2, Czech Republic
            University of British Columbia
            Author notes
            †Address correspondence to: David Staneˇk ( stanek@ 123456img.cas.cz ) and Petr Herman (herman@karlov.mff.cuni.cz).

            *These authors contributed equally to this work.

            Contributors
            Role: Monitoring Editor
            Journal
            Mol Biol Cell
            molbiolcell
            mbc
            Mol. Bio. Cell
            Molecular Biology of the Cell
            The American Society for Cell Biology
            1059-1524
            1939-4586
            15 February 2011
            : 22
            : 4
            : 513-523
            21177826
            3038649
            E10-07-0560
            10.1091/mbc.E10-07-0560
            (Monitoring Editor)
            © 2011 Novotný et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

            “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

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            Molecular biology

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