The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.
The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D002352:Carrier Proteins; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D011061:Poly A