Conformational changes of U6 RNA during the spliceosome cycle: an intramolecular helix is essential both for initiating the U4-U6 interaction and for the first step of slicing.
During each spliceosome cycle, U6 RNA undergoes several conformational changes, involving the formation and disruption of base-pairing interactions with U4 and U2 RNAs. By use of a mutational approach we have focused on the stem II region of U6, which can adopt alternative conformations: In the singular form of U6, it can form an intramolecular stem-loop structure; in the U4/U6 snRNP, the stem II region base-pairs with U4 RNA; in the active spliceosome, this region has been proposed to fold back into an intramolecular U6 helix in the context of a U6-U2 structure. Using chemical modification/interference assays and a mutational approach we found that the 3' terminal loop of the singular U6 (nucleotides 65-69) is essential for initiating the U4-U6 base-pairing interaction. A series of point mutations in the adjacent helix was designed to alter the stability of the intramolecular helix. Stabilizing mutations inhibited the formation of the U4/U6 snRNP. In contrast, mutant U6 RNAs with a destabilized intramolecular helix were still active in U4-U6 interaction and spliceosome assembly; however, their ability to support the first step of splicing was strongly reduced, suggesting that the intramolecular U6 helix has an important function in the first step of splicing. Affinity-purified U4 snRNP and U6 RNA did not assemble into a stable U4/U6 snRNP, unless complemented by nuclear extract, indicating that a protein factor (or factors) is necessary for the U4-U6 interaction. In sum, these data demonstrate that the stem II region of U6 functions both in U4-U6 interaction and in the first step of splicing; they also provide evidence that the balanced stability of different conformations of U6 RNA is critical for its function.
Wolff
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T
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Germany.
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D001483:Base Sequence; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012342:RNA, Small Nuclear