Identification of RNA cargoes by antibody-positioned RNA amplification.
The interactions between various RNA-binding proteins (RBPs) and the RNA transcripts they bind strongly influence posttranscriptional control of gene expression in vertebrates. The hundreds of vertebrate RBPs that have been identified within the genome, often with multiple RNA recognition motifs, are capable of recognizing specific target RNA sequences mediating the maturation, movement, and translational state of their RNA cargoes. To identify the cargoes associated with a specific RBP, we have developed a technique called antibody-positioned RNA amplification (APRA), which positions an oligonucleotide with a degenerate priming sequence in proximity to the RNAs sequestered by a specific RBP. The conjugation of the priming oligonucleotide to the antibody by itself does not interfere with the antibody's intrinsic affinity for the target RBP epitope, thus enabling RNA targets to be reverse-transcribed and amplified via a T7 bacteriophage RNA polymerase promoter sequence located upstream of the degenerate priming sequence in the oligonucleotide. By identifying the mRNA transcripts associated with the RBP in situ, we may be able to ascertain the significance of their temporal expression and physiological activities within the vast transcriptional networks regulating functional responses to stimuli.
Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Kevin Y
KY
Miyashiro
Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
James
J
Eberwine
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D017931:DNA Primers / Q000276:immunology / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D048348:Reverse Transcription; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D017931:DNA Primers; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases