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| Direct Observation Of The Molecular Splicing Process |
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| SciMed - Genetics & Genome | |||
| TS-Si News Service | |||
| Tuesday, 23 March 2010 15:00 | |||
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Ann Arbor, MI, USA. A research team has succeeded in making direct observations of the splicing process in single molecules. Humans are eukaryotes, an organism with cells that feature complex structures inside the membranes. Most eukaryotic genes are composed of numerous short coding sequences called exons, embedded within stretches of noncoding sequences called introns. A spliceosome is a complex of specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA (hnRNA) segment. By molecular-scale standards, the spliceosome is a monster of a machine, made up of five RNA and 100 or more protein subunits that agilely assemble, step-by-step, into the giant complex when it's time to carry out its work.
London, United Kingdom. New findings argue for the persistence of sex-linked chromosomes, such as the male Y  chromosome, refuting theories that the Y is doomed to extinction.
The results confirm that although these chromosom...The eventual goal is to construct a comprehensive model showing how RNA and the spliceosome can so faithfully interact throughout the splicing process to avoid the onset of malformations and disease. Close examination of the splicing process will help identify conditions that could lead to processig errors.Phillip A. Sharp was awarded the 1993 Nobel Prize in Medicine and Sharp subsequently discovered that after The resulting RNA molecule has the final transcript of instructions for the gene's protein-building activity. Since the initial discovery in 1977, gene splicing has been studied in a number of organisms, including yeast and human cells, using both genetic and biochemical approaches. While these methods can yield snapshots, they can't monitor the ongoing process. A team led by University of Michigan chemistry and biophysics professor Nils Walter, collaborating closely with a team led by internationally recognized splicing experts John Abelson and Christine Guthrie of the University of California, San Francisco, used an improved technique to observe the splicing process in single molecules. Their research appears in the journal Nature Structural and Molecular Biology.
The new study, which utilizes a technique called fluorescence resonance energy transfer (FRET) and a sophisticated microscope that watches single molecules in action, allows researchers to observe in real time the contortions involved in spliceosome assembly and operation. The spliceosome uses genetic material in RNA molecules to map out where to cut. RNA carries coded instructions for producing the proteins our body needs for building and repairing tissues, regulating body processes and many other sections called introns. The spliceosome's task is to recognize and excise introns which, once removed, can be stitched together by the spliceosome in various combinations. This mixing and matching of exons permits a relatively small number of genes (for humans, a little over 20,000) to serve as blueprints for a very large variety of proteins. Walter and his colleagues spied on the splicing process by attaching fluorescent tags to exons on either side of an intron in a short section of RNA they designed specifically for such studies. When laser light is shined on the tags, FRET can detect how close together or far apart the exons are. Repeated observations over time result in a molecular-scale movie that reveals how parts of the RNA molecule wiggle around, both before and during splicing. The researchers first studied the RNA in the absence of the spliceosome. "Conventional wisdom has been that the spliceosome directs the whole splicing process, that the RNA itself has little influence on it," Walter said. "But we saw the RNA molecule flexing on its own, with the intron folding and unfolding in a way that brings the exons closer together, suggesting a more active role for introns." When the team added an extract containing spliceosome components, along with ATP — the energy currency that fuels spliceosome assembly — the distance between exons first increased, then decreased even more, and splicing occurred. Interestingly, the series of contortions that RNA went through during splicing was not a one-way path; the steps were reversible. "Imagine the movie director having doubts about what scenes to cut and continuously going back and forth in holding different pieces of footage together before actually making a decision and splicing the film. That's what we saw happening at the molecular level," Walter said. "To our knowledge, our data provide the first direct glimpse of such reversible conformational changes during the splicing process." Next, the researchers plan to attach fluorescent tags to different parts of the system to see how the various parts relate to one another in space and time during splicing. The eventual goal is to construct a comprehensive model showing how RNA and the spliceosome can so faithfully interact throughout the splicing process to avoid the onset of malformations and disease. Close examination of the splicing process will help identify conditions that could lead to processig errors. FundingThe research was funded by the National Institutes of Health, the American Cancer Society, the Agouron Institute and the U-M Rackham Graduate School.
ParticipantsWalter's coauthors on the paper are U-M graduate students Mario Blanco, Franklin Fuller and Pavithra Aravamudhan; former graduate student Mark Ditzler; former undergraduate student Mona Wood; and Abelson, Guthrie, Tommaso Villa, Daniel Ryan, Jeffrey Pleiss and Corina Maeder of the University of California, San Francisco.
CitationConformational dynamics of single pre-mRNA molecules during
in vitro splicing. John Abelson, Mario Blanco, Mark A Ditzler, Franklin Fuller, Pavithra Aravamudhan, Mona Wood, Tommaso Villa, Daniel E Ryan, Jeffrey A Pleiss, Corina Maeder, Christine Guthrie and Nils G Walter. Nature Structural & Molecular Biology 2010; ePub ahead of print. doi:10.1038/nsmb.1767Abstract The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. During splicing in vitro, we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3'–splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.
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