Transcriptome-wide measurement of mRNA polyadenylation state.
The 3' poly(A) tail has important roles throughout the eukaryotic mRNA life cycle. A characteristic aspect of poly(A) tail function is furthermore that it can be modulated by changes in its length. This is in turn a well-recognised cellular means to regulate both, mRNA translation and stability, and a positive correlation has often been found between the efficiency of mRNA translation and the length of its poly(A) tail. Here we describe methodology to measure mRNA polyadenylation state in a transcriptome-wide manner, using separation of cellular mRNA populations on poly(U) sepharose in combination with microarray analysis of the resulting fractions. We further detail methods for bulk and mRNA-specific poly(A) tail length measurements to monitor the efficiency of initial mRNA separation and to verify candidates selected from the microarray data. Although detailed here for the study of yeast mRNAs, these methods are adaptable to the investigation of any cellular context in which poly(A) tail length control is known or suspected to operate.
Beilharz
Traude H
TH
Molecular Genetics Division, Victor Chang Cardiac Research Institute (VCCRI), Lowy Packer Building, 405 Liverpool Street, Darlinghurst (Sydney), NSW 2010, Australia. t.beilharz@victorchang.edu.au
Preiss
Thomas
T
D002850:Chromatography, Gel; D020869:Gene Expression Profiling / Q000379:methods*; D026723:Polyadenylation / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives
D012333:RNA, Messenger; C030188:poly U-sepharose; D012685:Sepharose