Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2013.
Splicing factor 1 (SF1) recognizes the consensus branch point sequence (BPS) of
the 3' splice site in the initial stages of spliceosome assembly. As I introduce in Chapter
1, a highly conserved SPSP motif within SF1 is predominately in the phosphorylated
state in mammalian cells. However, the structure and function of the phosphorylated SF1
SPSP-containing domain is poorly understood.
In Chapter 2, I report my crystal structure determination of the phosphorylated
SPSP-containing domain (P)SF114-132 bound to the C-terminal domain of U2AF65,
another essential splicing factor. The 2.29 Å resolution structure demonstrates that
phosphorylation induces a local disorder-to-order transition within a previously unknown
SF1/U2AF65 interface. Further, I show by small-angle X-ray scattering (SAXS) that the
local folding of the SPSP motif transduces into global conformational rearrangements in
the nearly full-length (P)SF1/U2AF65/ 3' splice site assembly. In Chapter 3, I describe my
small-angle X-ray scattering (SAXS) characterization of the nearly full-length or
truncated SF1/U2AF65 complexes in four states: apo-, phosphorylated, RNA-bound, and
phosphorylated/RNA-bound. The results indicate that the combination of SPSP
phosphorylation and association with the 3' splice site promotes a “C” shape for the
(P)SF1/U2AF65/RNA complex with the N-terminal domain of U2AF65 (RRM1) abutting
the C-terminal domain of SF1 (KH-QUA2).
Phosphorylation of the SF1 SPSP motif enhances U2AF65 interactions and
promotes formation of the ternary SF1/U2AF65/ 3' splice site complex. In Chapter 4, I use
isothermal titration calorimetry (ITC) to characterize the functional effect of SF1
phosphorylation on protein-protein interactions. The results suggest that rather than
directly promoting SF1/U2AF65 affinity, SF1 phosphorylation dictates U2AF65 ligand
specificity.
The two conserved serines within the SPSP motif are highly conserved and found
in the phosphorylated state in S. cerevisiae as well as humans. In the Chapter 5, I use a
genetic approach to investigate the functions of the phosphophorylated serines in S.
cerevisiae.
Altogether, this dissertation has provided a structural prototype for
phosphorylation-dependent control of pre-mRNA splicing factors and contributes to our
functional understanding of SF1 SPSP phosphorylation.
