Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.
A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes. Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.
Jandrositz
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A
Department of Biochemistry and Biophysics, University of California San Francisco 94143-0448.
Guthrie
C
C
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D011564:Furocoumarins / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D011485:Protein Binding; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry; D012342:RNA, Small Nuclear / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry*; D012441:Saccharomyces cerevisiae
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear