Ordered and Dynamic Assembly of Single Spliceosomes

被引：223

作者：

Hoskins, Aaron A. ^{[1
,3
]}

Friedman, Larry J. ^{[1
]}

Gallagher, Sarah S. ^{[2
]}

Crawford, Daniel J. ^{[1
,3
]}

Anderson, Eric G. ^{[3
]}

Wombacher, Richard ^{[2
]}

Ramirez, Nicholas ^{[3
]}

Cornish, Virginia W. ^{[2
]}

Gelles, Jeff ^{[1
]}

Moore, Melissa J. ^{[3
]}

机构：

[1] Brandeis Univ, Dept Biochem, Waltham, MA 02454 USA

[2] Columbia Univ, Dept Chem, New York, NY 10027 USA

[3] Univ Massachusetts, Howard Hughes Med Inst, Dept Biochem & Mol Pharmacol, Sch Med, Worcester, MA 01605 USA

来源：

SCIENCE | 2011年 / 331卷 / 6022期

关键词：

PRE-MESSENGER-RNA; MOLECULE FLUORESCENCE; YEAST SPLICEOSOME; CATALYTIC STEPS; EXON DEFINITION; IN-VIVO; COMPLEX; SNRNP; U1; MACHINE;

D O I：

10.1126/science.1198830

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

引用

页码：1289 / 1295

页数：7

共 23 条

[1] Conformational dynamics of single pre-mRNA molecules during in vitro splicing [J].

Abelson, John ;

Blanco, Mario ;

Ditzler, Mark A. ;

Fuller, Franklin ;

Aravamudhan, Pavithra ;

Wood, Mona ;

Villa, Tommaso ;

Ryan, Daniel E. ;

Pleiss, Jeffrey A. ;

Maeder, Corina ;

Guthrie, Christine ;

Walter, Nils G. .

NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2010, 17 (04) :504-U156

[2] RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition [J].

Bonnal, Sophie ;

Martinez, Concepcion ;

Foerch, Patrik ;

Bachi, Angela ;

Wilm, Matthias ;

Valcarcel, Juan .

MOLECULAR CELL, 2008, 32 (01) :81-95

[3] Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches [J].

Chen, Mo ;

Manley, James L. .

NATURE REVIEWS MOLECULAR CELL BIOLOGY, 2009, 10 (11) :741-754

[4] Visualizing the splicing of single pre-mRNA molecules in whole cell extract [J].

Crawford, Daniel J. ;

Hoskins, Aaron A. ;

Friedman, Larry J. ;

Gelles, Jeff ;

Moore, Melissa J. .

RNA, 2008, 14 (01) :170-179

[5] Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging [J].

Dave, Richa ;

Terry, Daniel S. ;

Munro, James B. ;

Blanchard, Scott C. .

BIOPHYSICAL JOURNAL, 2009, 96 (06) :2371-2381

[6] On/off blinking and switching behaviour of single molecules of green fluorescent protein [J].

Dickson, RM ;

Cubitt, AB ;

Tsien, RY ;

Moerner, WE .

NATURE, 1997, 388 (6640) :355-358

[7] KINETICS OF TERNARY COMPLEX-FORMATION BETWEEN DIHYDROFOLATE-REDUCTASE, COENZYME, AND INHIBITORS [J].

DUNN, SMJ ;

KING, RW .

BIOCHEMISTRY, 1980, 19 (04) :766-773

[8] The Evolutionarily Conserved Core Design of the Catalytic Activation Step of the Yeast Spliceosome [J].

Fabrizio, Patrizia ;

Dannenberg, Julia ;

Dube, Prakash ;

Kastner, Berthold ;

Stark, Holger ;

Urlaub, Henning ;

Luehrmann, Reinhard .

MOLECULAR CELL, 2009, 36 (04) :593-608

[9] Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence [J].

Friedman, Larry J. ;

Chung, Johnson ;

Gelles, Jeff .

BIOPHYSICAL JOURNAL, 2006, 91 (03) :1023-1031

[10] Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo [J].

Juillerat, A ;

Gronemeyer, T ;

Keppler, A ;

Gendreizig, S ;

Pick, H ;

Vogel, H ;

Johnsson, K .

CHEMISTRY & BIOLOGY, 2003, 10 (04) :313-317

← 1 2 3 →