Analysis of the structural determinants for RNA binding of the human protein AUF1/hnRNP D.
The protein AUF1/hnRNP D was one of the first factors identified that binds to the AU-rich region of certain mRNAs and mediates their fast degradation. Here we describe experiments to address the structural determinants for the binding of AUF1 to the RNA by combining comparative molecular modeling with gel shift assays. From our model of the RNA binding region of AUF1 we predicted that it interacts with RNA predominantly through stacking interactions that do not provide base-specific recognition. Only two RNA positions bound by AUF1 show base preferences: one for pyrimidine bases and the second for a conserved adenine residue. Gel shift assays with a panel of RNA oligonucleotides largely confirmed these model-based binding determinants. An alignment with proteins of the hnRNP family demonstrated that the amino acids involved in the stacking interactions are conserved whereas those that confer a base-specific recognition in AUF1 are variable.
Moraes
Karen C M
KC
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brasil.
Lee
Wen Hwa
WH
Kobarg
Jörg
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014169:Transformation, Bacterial / Q000235:genetics
C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D011993:Recombinant Fusion Proteins; D012313:RNA