Identification of oligoribonucleotide products of RNA cleavage in Saccharomyces cerevisiae
EMSL Project ID
2348
Abstract
We are proposing that mass spectrometry be used to identify, for the first time, small oligonucleotide RNAs that result from a specific enzymatic activity of RNA polymerase II in yeast. Newly synthesized RNAs are subject to hydrolysis by RNA polymerase II in a factor-dependent manner. This factor is a stimulatory protein that binds RNA polymerase and facilitates RNA chain elongation in an unusual manner; i.e. by stimulating an endoribonuclease activity of elongating RNA polymerase. This activity, which results in the generation of a specific 9 base RNA, has been well-studied in vitro and is ubiquitous across eukaryotic organisms. Intermediates or products of this activity have never been seen in vivo in any organism. The basic approach would be to generate a synthetic RNA that can serve as a standard for LC-MS. Extracts prepared from yeast will serve as experimental material. These extracts will be largely protein- and nucleic acid-free and should contain nucleotides and oligonucleotides. The use of yeast offers certain advantages in trying to identify a specific oligonucleotide product in a crude and complex mixture. We can use cells deleted of the gene (and therefore devoid of) the stimulatory factor that activates the nuclease. We can provide extrachromosomal DNA substrates that generate a large amount of specific substrate by placing specific plasmids within cells. Specific drug treatments and growth conditions of the cells should also modify the amount of substrate and product. Thus, any MS peak of interest should correlate with these other experimental manipulations. The largest perceived pitfall will likely be the positive identification of a specific oligonucleotide in a complex mixture that will no doubt contain many nucleotides and nucleotide derivatives. This is the rationale for using mass spectrometry.
Project Details
Project type
Exploratory Research
Start Date
2002-02-28
End Date
2003-08-27
Status
Closed
Released Data Link
Team
Principal Investigator