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Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins
Author(s) -
Nottrott Stephanie,
Urlaub Henning,
Lührmann Reinhard
Publication year - 2002
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdf544
Subject(s) - biology , small nuclear rna , computational biology , genetics , microbiology and biotechnology , evolutionary biology , rna , gene , rna dependent rna polymerase
During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6‐specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro . We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5′ portion of U4 snRNA via a novel RNA‐binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP‐specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.