The University of Southampton
University of Southampton Institutional Repository

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

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways
Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways
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
0305-1048
2051-2067
Chen, Li
63e9dd39-9be1-4416-82cb-58efdc1b23c1
Weinmeister, Robert
bb1f7d4b-1c66-4d63-a098-dbbc381ce7b2
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Eperon, Lucy P.
f7939c95-c6da-4af2-b911-ef4b36718513
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Hudson, Andrew J.
90cb2fdf-a316-46ae-98ca-d2e575ae58fc
Eperon, Ian C.
ded0b4c2-da7f-4de2-814b-13a30d0202ee
Chen, Li
63e9dd39-9be1-4416-82cb-58efdc1b23c1
Weinmeister, Robert
bb1f7d4b-1c66-4d63-a098-dbbc381ce7b2
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Eperon, Lucy P.
f7939c95-c6da-4af2-b911-ef4b36718513
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Hudson, Andrew J.
90cb2fdf-a316-46ae-98ca-d2e575ae58fc
Eperon, Ian C.
ded0b4c2-da7f-4de2-814b-13a30d0202ee

Chen, Li, Weinmeister, Robert, Kralovicova, Jana, Eperon, Lucy P., Vorechovsky, Igor, Hudson, Andrew J. and Eperon, Ian C. (2016) Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways. Nucleic Acids Research, 45 (4), 2051-2067. (doi:10.1093/nar/gkw860).

Record type: Article

Abstract

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

Text
2016 NAR stoichio.pdf - Version of Record
Available under License Creative Commons Attribution.
Download (1MB)

More information

Accepted/In Press date: 16 September 2016
e-pub ahead of print date: 28 September 2016
Organisations: Human Development & Health

Identifiers

Local EPrints ID: 401116
URI: https://eprints.soton.ac.uk/id/eprint/401116
ISSN: 0305-1048
PURE UUID: b709dd40-b5d1-43c7-9a50-e94578cf842a

Catalogue record

Date deposited: 07 Oct 2016 09:28
Last modified: 15 Jul 2019 20:02

Export record

Altmetrics

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×