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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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          Most cited references75

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          In vivo dynamics of RNA polymerase II transcription.

          We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min(-1), much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.
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            Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs.

            Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body-specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site-specific synthesis of 2'-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post-transcriptional modification of spliceosomal snRNAs.
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              Spliceosomal UsnRNP biogenesis, structure and function.

              Significant advances have been made in elucidating the biogenesis pathway and three-dimensional structure of the UsnRNPs, the building blocks of the spliceosome. U2 and U4/U6*U5 tri-snRNPs functionally associate with the pre-mRNA at an earlier stage of spliceosome assembly than previously thought, and additional evidence supporting UsnRNA-mediated catalysis of pre-mRNA splicing has been presented.
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                Author and article information

                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
                Affiliations
                [1] aInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic
                [2] bInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic
                [3] 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.

                Article
                E10-07-0560
                10.1091/mbc.E10-07-0560
                3038649
                21177826
                1d68745a-cdcf-47ee-9fa5-c5b6c39b24ce
                © 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.

                History
                : 02 July 2010
                : 08 December 2010
                : 15 December 2010
                Categories
                Articles
                Nuclear Functions
                A Highlights from MBoC Selection

                Molecular biology
                Molecular biology

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