Construction of 4-thiouridine site-specifically substituted RNAs for cross-linking studies.
Availability of 4-thiouridine (4-thioU)-containing RNAs is the prerequisite for 4-thioU site-specific cross-linking studies. This paper presents a method for constructing such RNAs. A 5'- and a 3'-RNA are synthesized via phage RNA polymerase transcription and/or RNase H site-specific cleavage directed by 2'-O-methyl-RNA-DNA chimeras. These two half-RNAs in combination correspond to the sequence of full-length RNA, with a single nucleotide gap at the junction that will be filled in with a 4-thiouridylate. A single p4SUp, which is derived from 4SUpN (N can be any nucleotide) via 5'-phosphorylation (therefore, the phosphate can be radioactive) followed by RNase A digestion, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3'-phosphate of the ligated product is subsequently removed by calf intestinal alkaline phosphatase to produce a 3'-hydroxyl group. The resulting 5'-half RNA and the 3'-half RNA with a 5'-phosphate group (which can also be radioactive) are then aligned with a bridging deoxyoligonucleotide and ligated with T4 DNA ligase. This method was previously applied to the P120 pre-mRNA that contains an AT-AC intron, yielding three RNAs each containing a single 4-thioU near the 5'-splice site. Subsequent cross-linking studies with these RNAs yielded detailed information regarding interactions between the 5'-splice site and other spliceosomal snRNAs and between the 5'-splice site and proteins during splicing. Because there is no sequence constraint surrounding the site of 4-thioU substitution, this method should be applicable to many other RNAs.
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Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, Connecticut 06536, USA.
D001435:Bacteriophages / Q000235:genetics; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D011088:DNA Ligases / Q000378:metabolism; D008956:Models, Chemical; D008957:Models, Genetic; D008967:Molecular Biology / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D013891:Thiouridine / Q000138:chemical synthesis* / Q000737:chemistry*; D013997:Time Factors
D003432:Cross-Linking Reagents; D013891:Thiouridine; D012313:RNA; D011088:DNA Ligases