Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus
Year: 2014
Type: article
Abstract: When isolated from mammalian cell nuclei, all nuclear pre-mRNAs are packaged in multi-subunit large ribonucleoprotein complexes-supraspliceosomes-composed of four native spliceosomes interconnected by the pre-mRNA. Supraspliceosomes contain all five spliceosomal U snRNPs, together with other splicing factors, and are functional in splicing. Supraspliceosomes studied thus far represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. To analyze specific splicing complexes, here, we affinity purified Pseudomonas aeruginosa phage 7 (PP7)-tagged splicing complexes assembled in vivo on Adenovirus Major Late (AdML) transcripts at specific functional stages, and characterized them using molecular techniques including mass spectrometry. First, we show that these affinity purified splicing complexes assembled on PP7-tagged AdML mRNA or on PP7-tagged AdML pre-mRNA are assembled in supraspliceosomes. Second, similar to the general population of supraspliceosomes, these defined supraspliceosomes populations are assembled with all five U snRNPs at all splicing stages. This study shows that dynamic changes in base-pairing interactions of U snRNA:U snRNA and U snRNA:pre-mRNA that occur in vivo during the splicing reaction do not require changes in U snRNP composition of the supraspliceosome. Furthermore, there is no need to reassemble a native spliceosome for the splicing of each intron, and rearrangements of the interactions will suffice.
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,499
Awards: Protein structures in the context of time and space by mass spectrometry., The Mammalian Cellular Splicing Machine - Structure and Function, Protein structures in the context of time and space by mass spectrometry., Three-dimensional proteomics: Mapping the geometry of large protein assemblies using cross-linking, mass spectrometry, and bioinformatics., Core funding renewal for the Wellcome Trust Centre for Cell Biology .
OpenAlex ID: W2000017815