Protocol

RNA-Protein Interaction Protocols

Volume 488 of the series Methods in Molecular Biology pp 357-365

Preparation of Efficient Splicing Extracts From Whole Cells, Nuclei, and Cytoplasmic Fractions

  • Naoyuki KataokaAffiliated withTokyo Medical and Dental University, Medical Research Institute
  • , Gideon DreyfussAffiliated withHoward Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine

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Summary

Pre-mRNA (messenger RNA) splicing is an essential step for gene expression in higher eukaryotes. Splicing reactions have been well studied in vitro using extracts prepared from cultured cells. We describe protocols for the preparation of splicing-competent extracts from whole cells, nuclei, and cytoplasmic fractions. The nuclear and whole-cell extracts are fully active in splicing, while S100 extracts are able to support splicing only when SR (Serine/Arginine-rich) proteins are supplied. The simple method described here to prepare splicing active extracts from whole cells is particularly useful in studying pre-mRNA splicing in many different cell types.

Key Words:

Nuclear extracts S100 extracts sonication splicing whole-cell extracts.