Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Author(s)

Carvalho, Felipe A. ; Barros, Moises R. A. ; Girotto, Thierry P. F. ; Perona, M. Griselda ; Oliveira, Carla C.

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 165, p. 9-pg., 2020-11-01.

Abstract

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors. (AU)

• FAPESP's process: 15/06477-9 - Functional study of the components of the Saccharomyces cerevisiae exosome and the spliceosome, and their role in posttranscriptional control of gene expression
• Grantee: Carla Columbano de Oliveira
• Support Opportunities: Research Projects - Thematic Grants