Isolation of mRNAs encoding peroxisomal proteins from yeast using a combined cell fractionation and affinity purification procedure

Abstract

Targeted mRNA localization to distinct subcellular sites occurs throughout the eukaryotes and presumably allows for the localized translation of proteins near their site of function. Specific mRNAs have been localized in cells using a variety of reliable methods, such as fluorescence in situ hybridization with labeled RNA probes, mRNA tagging using RNA aptamers and fluorescent proteins that recognize these aptamers, and quenched fluorescent RNA probes that become activated upon binding to mRNAs. However, fluorescence-based RNA localization studies can be strengthened when coupled with cell fractionation and membrane isolation techniques in order to identify mRNAs associated with specific organelles or other subcellular structures. Here we describe a novel method to isolate mRNAs associated with peroxisomes in the yeast, Saccharomyces cerevisiae. This method employs a combination of density gradient centrifugation and affinity purification to yield a highly enriched peroxisome fraction suitable for RNA isolation and reverse transcription-polymerase chain reaction detection of mRNAs bound to peroxisome membranes. The method is presented for the analysis of peroxisome-associated mRNAs; however it is applicable to studies on other subcellular compartments.