ArticleMethods in molecular biology (Clifton, N.J.)2023
Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway.
Article in Methods in molecular biology (Clifton, N.J.), 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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1 citing paper in PubMed.
- A Preliminary Exploration of Transcriptome and Proteomic Changes During the Young and Harvest Periods inJournal of fungi (Basel, Switzerland) · 2025Article
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2 authors.
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Abstract
The spliceosome is a dynamic ribonucleoprotein particle and is assembled via sequential binding of five snRNAs and numerous protein factors. To understand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free splicing extracts, and in vitro splicing reactions, as well as procedures to arrest the spliceosome at different stages of the pathway for characterization of specific splicing complexes from the budding yeast Saccharomyces cerevisiae. Methods for arresting spliceosomes at specific stages include depletion with antibodies against factors required for specific steps of the pathway, use of extracts prepared from temperature-sensitive mutants, use of dominant negative mutants of DExD/H-box proteins, and use of mutant substrates.
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