A new strategy for introducing photoactivatable 4-thiouridine ((4S)U) into specific positions in a long RNA molecule.
We describe a new protocol, which does not require (4S)UpG, for introducing (4S)U into specific sites in a pre-mRNA substrate. A 5'-half and a full-length RNA are first synthesized by phage RNA polymerase. p(4S)Up, which is derived from (4S)UpU and can therefore be 32P-labeled, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3' phosphate of the ligated product is removed subsequently by CIP (calf intestinal alkaline phosphatase) to produce a 3'-OH group. The 3'-half RNA with a 5' phosphate is produced by site-specific RNase H cleavage of the full-length pre-mRNA directed by a 2'-O-methyl RNA-DNA chimera. The two half RNAs are then aligned with a bridging oligonucleotide and ligated with T4 DNA ligase. Our results show that 32P-p(4S)Up ligation to the 3' end of the 5'-half RNA is comparable to 32P-pCp ligation. Also, the efficiency of the bridging oligonucleotide-mediated two-piece ligation is quite high, approximately 30-50%. This strategy has been applied to the P120 pre-mRNA containing an AT-AC intron, but should be applicable to many other RNAs.
Yu
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YT
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Steitz
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JA
D000345:Affinity Labels / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D013891:Thiouridine / Q000737:chemistry*; D014158:Transcription, Genetic
D000345:Affinity Labels; D013891:Thiouridine; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H