Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.
Article Details
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Jurica MS, Licklider LJ, Gygi SR, Grigorieff N, Moore MJ
Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.
RNA. 2002 Apr;8(4):426-39.
- PubMed ID
- 11991638 [ View in PubMed]
- Abstract
We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.
DrugBank Data that Cites this Article
- Polypeptides
Name UniProt ID Heterogeneous nuclear ribonucleoproteins A2/B1 P22626 Details Heterogeneous nuclear ribonucleoprotein H P31943 Details Splicing factor 3A subunit 2 Q15428 Details Splicing factor 3B subunit 3 Q15393 Details Heterogeneous nuclear ribonucleoprotein F P52597 Details Heterogeneous nuclear ribonucleoprotein K P61978 Details Splicing factor 3A subunit 3 Q12874 Details Small nuclear ribonucleoprotein Sm D2 P62316 Details Serine/arginine-rich splicing factor 1 Q07955 Details Probable ATP-dependent RNA helicase DDX5 P17844 Details Splicing factor 3B subunit 1 O75533 Details Serine/threonine-protein kinase PRP4 homolog Q13523 Details