Structure and function of steroid receptor RNA activator protein, the proposed partner of SRA noncoding RNA.
In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 10(5) molecules per cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein-protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.
BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.|Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.|Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA.
David B
DB
McKay
BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.|Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Linghe
L
Xi
BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.|Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Kristen K B
KKB
Barthel
BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.|Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.|Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.|Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA.
Thomas R
TR
Cech
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D040681:Structural Homology, Protein
D002352:Carrier Proteins; C080779:PRP18 protein, S cerevisiae; D022661:RNA, Untranslated; D011994:Recombinant Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C548898:steroid receptor RNA activator, human; D004958:Estradiol
RNA-binding protein; estrogen; nuclear receptor; steroid receptor activator; transcription factor