Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system.

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.

journal_name

Genes Dev

journal_title

Genes & development

authors

Johnson TL,Abelson J

doi

10.1101/gad.895601

subject

Has Abstract

pub_date

2001-08-01 00:00:00

pages

1957-70

issue

15

eissn

0890-9369

issn

1549-5477

journal_volume

15

pub_type

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