In vivo, high-resolution analysis of yeast and mammalian RNA-protein interactions, RNA structure, RNA splicing and ribozyme cleavage by use of terminal transferase-dependent PCR.
We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin [J. Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)]. When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step. The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer. This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR. Band patterns obtained are reproducible and quantifiable. We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage. Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.
Chen
H H
HH
Departments of Biology and Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Castanotto
D
D
LeBon
J M
JM
Rossi
J J
JJ
Riggs
A D
AD
D001483:Base Sequence; D002460:Cell Line; D004253:DNA Nucleotidylexotransferase; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012313:RNA; D004253:DNA Nucleotidylexotransferase