An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.uring gene expression, the removal of introns is essential for translation of mature mRNA. The splicing process involves a large number of splicing factors for the correct recognition of introns (1). Whereas U1 snRNP contacts the 5′ splice site, recognition of the 3′ splice site involves binding of SF1/BBP to the branch point sequence (BPS) (2-5) and binding of the U2 auxiliary factor (U2AF) heterodimer to the poly-pyrimidine-tract (Py-tract) that precedes the AG dinucleotide at the intron/exon junction. Binding of U2AF to the 3′ splice site during the early steps of spliceosome assembly recruits the U2 snRNP (6-9). The strength, i.e., splicing efficiency, of a 3′ splice site requires recognition of the BPS, Py-tract, and the AG dinucleotide. However, of these three RNA elements, the Py-tract exhibits the largest degree of variability, and thus, weak to strong splice sites are primarily classified depending on the composition of the Py-tract (7, 10).U2AF is a heterodimer consisting of a large (U2AF65) and a small (U2AF35) subunit. U2AF65 harbors two canonical RNA recognition motifs (RRM1,2) and an atypical C-terminal RRM domain, called the U2AF homology motif (UHM). U2AF35 has one RRM (which acts as an UHM), flanked N-and C-terminally by two CCCH-type zinc finger motifs, respectively (Fig. S1A) (9, 11, 12). The U2AF heterodimer is formed by recognition of a peptide motif, cal...

Fluorescence-based techniques are widely used to study biomolecular conformations, intra- and intermolecular interactions, and conformational dynamics of macromolecules. Especially for fluorescent-based single-molecule experiments, the choice of the fluorophore and labeling position are highly important. Here, we have studied the biophysical and structural effects that are associated with the conjugation of fluorophores to cysteines in the splicing factor U2AF65 by using single pair Förster resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) spectroscopy. We show that certain acceptor fluorophores are advantageous depending on the experiments performed. The effects of dye attachment on the protein conformation were characterized using heteronuclear NMR experiments. We find that the presence of hydrophobic and aromatic moieties in the fluorophores can significantly affect the conformation of the conjugated protein, presumably by transient interactions with the protein surface. Guidelines are provided for carefully choosing fluorophores, considering their photophysical properties and chemical features for the design of FRET experiments and for minimizing artifacts.