U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.
U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.
D000818:Animals; D017164:Ascaris lumbricoides / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D013329:Structure-Activity Relationship; D013873:Thionucleotides
D012322:RNA Precursors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D013873:Thionucleotides