Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding.
The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential splicing factor U2AF(65), which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF(65) shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF(65) and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.
Banerjee
Hiren
H
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, 80309, USA.
Rahn
Andrew
A
Davis
William
W
Singh
Ravinder
R
D001483:Base Sequence; D001665:Binding Sites; D029721:Drosophila Proteins / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D012313:RNA / Q000737:chemistry*; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*
D029721:Drosophila Proteins; D011743:Pyrimidines; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C067777:Sxl protein, Drosophila; D012313:RNA; C030986:pyrimidine