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-dep endent 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 extremel y sensitive, versatile, quantitative and nucleotide-level assay for detecti ng 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 deoxynucleo tidyl 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 neste d primers and the sensitivity of PCR. Band patterns obtained are reproducib le and quantifiable. We successfully used the technique for the study of ye ast RNA structure, splicing intermediates and ribozyme cleavage. Also, in v ivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elem ents in the mRNAs of transferrin receptor and ferritin H-chain.