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

Li Chen,Ian C Eperon,Jana Kralovicova,Igor Vorechovsky,Robert Weinmeister,Andrew J Hudson,Lucy P Eperon

doi:10.1093/nar/gkw860

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.

Highlights

The processes of recognition and selection of 3 splice sites (3 ss) are complex and poorly understood. 3 ss comprise several distinguishable elements that are recognized directly by RNA-binding proteins: the branch site, a polypyrimidine-rich tract and an AG preceding the 3 ss itself
Nuclear extracts were prepared from HEK293T cells transfected with plasmids expressing either mEGFP-U2B, mEGFP-SF3A3 or both mEGFP-U2AF65 and mCherry-U2AF35; these were pre-incubated for 15 min in the presence or absence of adenosine triphosphate (ATP) and phosphocreatine, pre-mRNA was added to 62.5 nM and reactions were incubated, as described [64]
To enable the binding of individual molecules of U2AF65 and U2AF35 to pre-mRNA in nuclear extracts to be detected, U2AF65 and U2AF35 were co-expressed as Nterminal fusions with mEGFP and mCherry respectively

Summary

Introduction

The processes of recognition and selection of 3 splice sites (3 ss) are complex and poorly understood. 3 ss comprise several distinguishable elements that are recognized directly by RNA-binding proteins: the branch site, a polypyrimidine-rich tract and an AG preceding the 3 ss itself. U2AF65 associates with U2AF35 as a stable heterodimer [1], and interacts with SF1/mBBP [8,9], which recognizes the branch site but is not an essential splicing factor [10,11,12]. The U2 snRNP is recruited later by interactions with SF1 and U2AF65, and base-pairs with the pre-mRNA around the branch site, displacing SF1 [12]. It is not clear whether any or all of these sequence-specific interactions with the pre-mRNA occur before or after the transition from recognition to selection of the 3 ss. Only one molecule of factors recruited after selection would associate per intron, regardless of the strength of alternative sites

Objectives

Methods

Results

Conclusion