Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein

Abstract

In yeast (Saccharoymces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated.

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

“…Comparison to SF1 highlights key differences in the secondary structure, position, and amino acid sequence of the QUA2 module that help rationalize the different stringencies of recognition of the U 1 A 2 C 3 nucleobases. These structural insights are in keeping with biochemical studies of chimeric Msl5-SF1 proteins that implicated the QUA2 module, and its Lys252-Arg253 dipeptide, as determinants of Msl5 target specificity and affinity in vitro (Garrey et al 2008). Yet, this dipeptide is clearly not decisive per se for Msl5 function in vivo, insofar as a yeast MSL5-(K252A-R253A) strain grows as well as wild-type MSL5 cells at 25°C-37°C .…”

“…Lys252 also donates a hydrogen bond to the cytosine O2 carbonyl of the C 3 nucleobase. These features of the crystal structure neatly explain the results of Garrey et al (2008), who reported that the Lys252-Arg253 dipeptide of yeast Msl5 (which differs from the corresponding Arg240-Lys241 dipeptide in human SF1) is a key determinant of the distinctive high RNA affinity and sequence specificity of Msl5 versus SF1. The NMR structure of SF1 was not instructive on this front, insofar as the SF1 Arg240 residue is remote from the RNA (Liu et al 2001).…”

“…Msl5 and human SF1 are orthologous branchpoint-binding splicing factors with distinctive site specificities and affinities that correlate with the conservation (in yeast) or diversity (in humans) of native intron branchpoint sequences. Whereas Msl5 is sensitive to perturbations of the U 1 A 2 C 3 and C 7 nucleobases of the consensus branchpoint, SF1 is not (Berglund et al 1997;Garrey et al 2008). The NMR structure of SF1 bound to a yeast consensus branchpoint RNA illuminated the principles of RNA recognition by KH-QUA2 proteins (Liu et al 2001), but not the basis for the functional differences between the yeast and human orthologs.…”

“…Also, in the view shown in Figure 4, the QUA2 element is displaced downward in SF1 compared with its position in Msl5 and the two other KH-QUA2 proteins. These structural differences might explain prior findings, based on studies with chimeric yeast-human branchpoint binding proteins, that the QUA2 domain dictates the higher RNA affinity and sequence specificity of yeast Msl5 versus human SF1 (Garrey et al 2008). …”

Insights into the Structural Basis of RNA Recognition by Star Domain Proteins

“…Also, two basic amino acids within the QUA2 domain (R240 and K241) that have been experimentally demonstrated to contribute to binding affinity and specificity without directly contacting RNA are not conserved in Sam68, but are in GLD-1, QKI, and HOW. 66 Together, the evidence suggests that the Sam68 QUA2 domain diverges significantly from the others, and as such its role in RNA recognition is not clear.…”