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Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

journal contribution
posted on 2016-12-02, 11:45 authored by Li Chen, Robert Weinmeister, J. Kralovicova, Lucy P. Eperon, I. Vorechovsky, Andrew J. Hudson, Ian C. Eperon
The selection of 3' splice sites (3'ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3'ss. Pre-mRNA molecules with two alternative 3'ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5' and 3' sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

Funding

Bloodwise [12060 to I.V.].

History

Citation

Nucleic Acids Research, 2016

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Old Departments Pre 01 Aug 2015/Department of Biochemistry (Pre 01 Aug 2015)

Version

  • VoR (Version of Record)

Published in

Nucleic Acids Research

Publisher

Oxford University Press (OUP)

issn

0305-1048

eissn

1362-4962

Acceptance date

2016-09-16

Copyright date

2016

Available date

2016-12-02

Publisher version

http://nar.oxfordjournals.org/content/early/2016/09/28/nar.gkw860.abstract

Notes

Supplementary Data are available at NAR Online and attached to this record.

Language

en

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