Publication Date:
2011-03-12
Description:
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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086749/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086749/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hoskins, Aaron A -- Friedman, Larry J -- Gallagher, Sarah S -- Crawford, Daniel J -- Anderson, Eric G -- Wombacher, Richard -- Ramirez, Nicholas -- Cornish, Virginia W -- Gelles, Jeff -- Moore, Melissa J -- F32 GM079971/GM/NIGMS NIH HHS/ -- F32 GM079971-03/GM/NIGMS NIH HHS/ -- GM079971/GM/NIGMS NIH HHS/ -- GM759628/GM/NIGMS NIH HHS/ -- K99 GM086471/GM/NIGMS NIH HHS/ -- K99 GM086471-02/GM/NIGMS NIH HHS/ -- K99/R00 GM086471/GM/NIGMS NIH HHS/ -- R01 GM043369/GM/NIGMS NIH HHS/ -- R01 GM053007/GM/NIGMS NIH HHS/ -- R01 GM053007-15/GM/NIGMS NIH HHS/ -- R01 GM081648/GM/NIGMS NIH HHS/ -- R01 GM081648-04/GM/NIGMS NIH HHS/ -- R01 GM54469/GM/NIGMS NIH HHS/ -- R01 GM81648/GM/NIGMS NIH HHS/ -- R37 GM043369/GM/NIGMS NIH HHS/ -- R37 GM043369-21/GM/NIGMS NIH HHS/ -- RC1 GM091804/GM/NIGMS NIH HHS/ -- RC1 GM091804-02/GM/NIGMS NIH HHS/ -- T32 GM007596/GM/NIGMS NIH HHS/ -- T32 GM007596-30/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2011 Mar 11;331(6022):1289-95. doi: 10.1126/science.1198830.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21393538" target="_blank"〉PubMed〈/a〉
Keywords:
Adenosine Triphosphate/metabolism
;
Fluorescent Dyes
;
Introns
;
Kinetics
;
Microscopy, Fluorescence
;
Protein Binding
;
RNA Precursors/*metabolism
;
*RNA Splicing
;
RNA, Fungal/*metabolism
;
Ribonucleoprotein, U1 Small Nuclear/metabolism
;
Ribonucleoprotein, U2 Small Nuclear/metabolism
;
Ribonucleoprotein, U4-U6 Small Nuclear/metabolism
;
Ribonucleoprotein, U5 Small Nuclear/metabolism
;
Ribonucleoproteins, Small Nuclear/*metabolism
;
Saccharomyces cerevisiae/genetics/*metabolism/ultrastructure
;
Saccharomyces cerevisiae Proteins/*metabolism
;
Spliceosomes/*metabolism
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
