RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.
BACKGROUND
The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.


RESULTS
We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.


CONCLUSIONS
We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.
López de Heredia
Miguel
M
Gene Centre and Institute for Biochemistry, University of Munich, Feodor Lynen Str, 25, D-81377 Munich, Germany. lopez@lmb.uni-muenchen.de
Jansen
Ralf-Peter
RP
D002458:Cell Fractionation / Q000379:methods / Q000592:standards; D004791:Enzyme Inhibitors / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012331:RNA, Fungal; D012260:Ribonucleases