Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site
Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy tr...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2013-04, Vol.110 (17), p.6783-6788 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Crawford, Daniel J. Hoskins, Aaron A. Friedman, Larry J. Gelles, Jeff Moore, Melissa J. |
| description | Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5′ splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites. |
| doi_str_mv | 10.1073/pnas.1219305110 |
| format | Article |
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We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5′ splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. 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Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.</description><subject>Base Sequence</subject><subject>Biological Sciences</subject><subject>Catalysis</subject><subject>DNA Primers - genetics</subject><subject>Dyes</subject><subject>Fluorescence</subject><subject>Image Processing, Computer-Assisted</subject><subject>Introns</subject><subject>Messenger RNA</subject><subject>Microscopy, Fluorescence</subject><subject>Molecular Sequence Data</subject><subject>Molecules</subject><subject>Nucleic Acid Conformation</subject><subject>Oligonucleotides - genetics</subject><subject>Proteins</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA Splice Sites - genetics</subject><subject>RNA Splicing - physiology</subject><subject>Saccharomyces cerevisiae</subject><subject>Small nuclear ribonucleoproteins</subject><subject>Small nuclear RNA</subject><subject>Spectrum Analysis</subject><subject>Spliceosomes</subject><subject>Spliceosomes - physiology</subject><subject>Splicing</subject><subject>Yeasts</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkcFu1DAQhi0EokvhzAmw1AuXtJ44tuNLJVS1gFQJibZny3GcXa-8cbCTleDEpS_EI_EkOOyyBU6WNd98nvGP0Esgp0AEPRt6nU6hBEkJAyCP0AKIhIJXkjxGC0JKUdRVWR2hZymtCSGS1eQpOiop47KsYYHub1y_9LbYBG_N5C02wQejvfumRxd6fPX58hbbrWttbyweV3rEafDO2JDCxmJtRrfdkUO0xrY24TTqJov0MMSgzQqHDrOf33_s-3ByYy72LW6i7nN5vj9HTzrtk32xP4_R3dXl7cWH4vrT-48X764LU0kYCykrWpG6KUG31kDb5D0pr0RnBMiusZKZTrSGtx2VdUtYyUoCHISpgXfQNfQYne-8w9RsbGtsP0bt1RDdRsevKmin_q30bqWWYasop0JQlgVv94IYvkw2jWrjkrHe696GKSmgFQcQNS8zevIfug5T7PN6vykBDOQsPNtRJoaUou0OwwBRc8Rqjlg9RJw7Xv-9w4H_k2kG3uyBufOgm31CcVHTTLzaEes0hnhAqpLJ_AZ_MHQ6KL2MLqm7m_krCQHKGeH0Fx68wms</recordid><startdate>20130423</startdate><enddate>20130423</enddate><creator>Crawford, Daniel J.</creator><creator>Hoskins, Aaron A.</creator><creator>Friedman, Larry J.</creator><creator>Gelles, Jeff</creator><creator>Moore, Melissa J.</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20130423</creationdate><title>Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site</title><author>Crawford, Daniel J. ; 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| subjects | Base Sequence Biological Sciences Catalysis DNA Primers - genetics Dyes Fluorescence Image Processing, Computer-Assisted Introns Messenger RNA Microscopy, Fluorescence Molecular Sequence Data Molecules Nucleic Acid Conformation Oligonucleotides - genetics Proteins Ribonucleic acid RNA RNA Splice Sites - genetics RNA Splicing - physiology Saccharomyces cerevisiae Small nuclear ribonucleoproteins Small nuclear RNA Spectrum Analysis Spliceosomes Spliceosomes - physiology Splicing Yeasts |
| title | Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site |
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