Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

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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
NameUniProt ID
Heterogeneous nuclear ribonucleoproteins A2/B1P22626Details
Heterogeneous nuclear ribonucleoprotein HP31943Details
Splicing factor 3A subunit 2Q15428Details
Splicing factor 3B subunit 3Q15393Details
Heterogeneous nuclear ribonucleoprotein FP52597Details
Heterogeneous nuclear ribonucleoprotein KP61978Details
Splicing factor 3A subunit 3Q12874Details
Small nuclear ribonucleoprotein Sm D2P62316Details
Serine/arginine-rich splicing factor 1Q07955Details
Probable ATP-dependent RNA helicase DDX5P17844Details
Splicing factor 3B subunit 1O75533Details
Serine/threonine-protein kinase PRP4 homologQ13523Details