Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3' poly(A) end phenotype.
The use of DNA microarrays has revolutionized the manner in which mRNA populations are analyzed. One limitation of the current technology is that mRNAs are often purified on the basis of their 3' poly(A) ends, which can be extremely short or absent in some mRNAs. To circumvent this limitation, we have developed a procedure for the purification of eukaryotic mRNAs using a mutant version of the mRNA 5' cap-binding protein (eIF4E) with increased affinity for the m7GTP moiety of the cap. By using this procedure, we have compared the populations of mammalian mRNAs purified by oligo(dT) and 5' cap selection with oligonucleotide microarrays. This analysis has identified a subpopulation of mRNAs that are present with short 3' poly(A) ends at steady state and are missed or underrepresented after purification by oligo(dT). These mRNAs may respond to specific posttranscriptional control mechanisms such as cytoplasmic polyadenylation.
Choi
Youkyung Hwang
YH
Department of Medicine, Genetics Program, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.
Hagedorn
Curt H
CH
D001483:Base Sequence; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D012315:RNA Caps / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics
D039561:Eukaryotic Initiation Factor-4E; D009838:Oligodeoxyribonucleotides; D012315:RNA Caps; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C027903:oligo (dT)