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Dynamics and consequences of spliceosome E complex formation.

ABSTRACT: The spliceosome must identify the correct splice sites (SS) and branchsite (BS) used during splicing. E complex is the earliest spliceosome precursor in which the 5' SS and BS are defined. Definition occurs by U1 small nuclear ribonucleoprotein (snRNP) binding the 5' SS and recognition of the BS by the E complex protein (ECP) branchpoint bridging protein (BBP). We have used single molecule fluorescence to study Saccharomyces cerevisiae U1 and BBP interactions with RNAs. E complex is dynamic and permits frequent redefinition of the 5' SS and BS. BBP influences U1 binding at the 5' SS by promoting long-lived complex formation. ECPs facilitate U1 association with RNAs with weak 5' SS and prevent U1 accumulation on RNAs containing hyperstabilized 5' SS. The data reveal a mechanism for how U1 binds the 5' SS and suggest that E complex harnesses this mechanism to stimulate recruitment and retention of U1 on introns.

SUBMITTER: Larson JD

PROVIDER: S-EPMC5779234 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Dynamics and consequences of spliceosome E complex formation.

Larson Joshua Donald JD Hoskins Aaron A AA

eLife 20170822

The spliceosome must identify the correct splice sites (SS) and branchsite (BS) used during splicing. E complex is the earliest spliceosome precursor in which the 5' SS and BS are defined. Definition occurs by U1 small nuclear ribonucleoprotein (snRNP) binding the 5' SS and recognition of the BS by the E complex protein (ECP) branchpoint bridging protein (BBP). We have used single molecule fluorescence to study <i>Saccharomyces cerevisiae</i> U1 and BBP interactions with RNAs. E complex is dynam ...[more]

PMID: 28829039

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Project description:Limited proteolysis of RNase-A yields a short N-terminal S-peptide segment and the larger S-protein. Binding of S-peptide to S-protein results in the formation of an enzymatically active RNase-S protein. S-peptide undergoes a transition from intrinsic disorder to an ordered helical state upon association with S-protein to form RNase-S and is an excellent model system to study coupled folding and binding. To better understand the dynamics of the RNases-S complex and its isolated partners, comparative molecular dynamics simulations have been performed. In agreement with experiment, we find significant conformational fluctuations of the isolated S-peptide compatible with a disordered regime and only little residual helical structure. In the RNase-S complex, the N-terminal helix of S-peptide unfolds and refolds repeatedly on the microsecond timescale, indicating that the ?-helical structure is only part of the equilibrium regime for these residues whereas the C-terminal residues are confined to the helical conformation that is found in the x-ray structure. This is also in line with systematic, in silico Alanine scanning free-energy simulations, which indicate that the major contribution to complex stability emerges from the C-terminal helical turn, consisting of residues 8-13 in S-peptide whereas the N-terminal S-peptide residues 1-7 make only minor contributions. Comparative simulations of S-protein in the presence and absence of S-peptide reveal that the isolated S-protein is significantly more flexible than in the complex, and undergoes a global pincerlike conformational change that narrows the S-peptide binding cleft. The narrowed binding cleft adds a barrier for complex formation likely influencing the binding kinetics. This conformational change is reversed by S-peptide association, which also stabilizes conformational fluctuations in S-protein. Such global motions associated with binding are also likely to play a role for other coupled peptide folding and binding processes at peptide binding regions on protein surfaces.

Dataset Information

Dynamics and consequences of spliceosome E complex formation.

Publications

Dynamics and consequences of spliceosome E complex formation.

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