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Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system.
Genes Dev. 2001 Aug 01; 15(15):1957-70.GD

Abstract

Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA.

Authors+Show Affiliations

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11485990

Citation

Johnson, T L., and J Abelson. "Characterization of U4 and U6 Interactions With the 5' Splice Site Using a S. Cerevisiae in Vitro Trans-splicing System." Genes & Development, vol. 15, no. 15, 2001, pp. 1957-70.
Johnson TL, Abelson J. Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system. Genes Dev. 2001;15(15):1957-70.
Johnson, T. L., & Abelson, J. (2001). Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system. Genes & Development, 15(15), 1957-70.
Johnson TL, Abelson J. Characterization of U4 and U6 Interactions With the 5' Splice Site Using a S. Cerevisiae in Vitro Trans-splicing System. Genes Dev. 2001 Aug 1;15(15):1957-70. PubMed PMID: 11485990.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system. AU - Johnson,T L, AU - Abelson,J, PY - 2001/8/4/pubmed PY - 2001/9/14/medline PY - 2001/8/4/entrez SP - 1957 EP - 70 JF - Genes & development JO - Genes Dev VL - 15 IS - 15 N2 - Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA. SN - 0890-9369 UR - https://www.unboundmedicine.com/medline/citation/11485990/Characterization_of_U4_and_U6_interactions_with_the_5'_splice_site_using_a_S__cerevisiae_in_vitro_trans_splicing_system_ L2 - http://www.genesdev.org/cgi/pmidlookup?view=long&pmid=11485990 DB - PRIME DP - Unbound Medicine ER -