Abstract
The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.
Highlights
Nals or splicing factors [3,4,5]
The U2AF65 subunit serves as an extensive molecular surface, with domains responsible for critical functions (Fig. 1A). (i) As addressed here, two RNA recognition motifs (RRMs) recognize the Py tract splice site signal [11, 12]; (ii) a region near the RRMs recruits the ATPase UAP56 to the assembling spliceosome [13]; (iii) a C-terminal U2AF homology motif (UHM) domain organizes the SF1 [14] and SF3b155 [15] splicing factors at the branch point sequence; (iv) a tryptophan-containing UHM ligand motif [16] positions the U2AF35 small subunit at the 3Ј splice site [17]; and (v) an N-terminal arginine-serine-rich (RS) domain promotes branch point sequence/U2 small nuclear RNA annealing [18]
The overall structures for U2AF65 and its 3Ј splice site assemblies are currently unknown, which presents a major obstacle to understanding their critical role during initiation of pre-mRNA splicing
Summary
Nals or splicing factors [3,4,5]. The splicing machinery (spliceosome) is faced with the task of recognizing relatively short exons (ϳ150 nucleotides on average in the human genome) located within vast stretches of intron RNA (Ͼ1500 nucleotides) [6]. (i) As addressed here, two RNA recognition motifs (RRMs) recognize the Py tract splice site signal [11, 12]; (ii) a region near the RRMs recruits the ATPase UAP56 to the assembling spliceosome [13]; (iii) a C-terminal U2AF homology motif (UHM) domain organizes the SF1 [14] and SF3b155 [15] splicing factors at the branch point sequence; (iv) a tryptophan-containing UHM ligand motif [16] positions the U2AF35 small subunit at the 3Ј splice site [17]; and (v) an N-terminal arginine-serine-rich (RS) domain promotes branch point sequence/U2 small nuclear RNA annealing [18]. These results support a another in the context of the folded three-dimensional struc- model whereby the U2AF65 RRM1 and RRM2 domains act with ture, possibly by a bent configuration of the central RRMs
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