Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60
Abstract
Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work definesmore »
- Authors:
-
- Yale Univ., New Haven, CT (United States)
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE; American Cancer Society (ACS)
- OSTI Identifier:
- 1736295
- Grant/Contract Number:
- RSG-0-222-01
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 15; Journal Issue: 11; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; RNA structure; hydrogen bonding; crystal structure; nucleic acids; uracils; dimerization; nucleotides; nucleotide mapping
Citation Formats
Hsiao, Hsin-hao T., Crichlow, Gregg V., Murphy, James W., Folta-Stogniew, Ewa J., Lolis, Elias J., and Braddock, Demetrios T. Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60. United States: N. p., 2020.
Web. doi:10.1371/journal.pone.0242725.
Hsiao, Hsin-hao T., Crichlow, Gregg V., Murphy, James W., Folta-Stogniew, Ewa J., Lolis, Elias J., & Braddock, Demetrios T. Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60. United States. https://doi.org/10.1371/journal.pone.0242725
Hsiao, Hsin-hao T., Crichlow, Gregg V., Murphy, James W., Folta-Stogniew, Ewa J., Lolis, Elias J., and Braddock, Demetrios T. Mon .
"Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60". United States. https://doi.org/10.1371/journal.pone.0242725. https://www.osti.gov/servlets/purl/1736295.
@article{osti_1736295,
title = {Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60},
author = {Hsiao, Hsin-hao T. and Crichlow, Gregg V. and Murphy, James W. and Folta-Stogniew, Ewa J. and Lolis, Elias J. and Braddock, Demetrios T.},
abstractNote = {Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.},
doi = {10.1371/journal.pone.0242725},
journal = {PLoS ONE},
number = 11,
volume = 15,
place = {United States},
year = {Mon Nov 30 00:00:00 EST 2020},
month = {Mon Nov 30 00:00:00 EST 2020}
}
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