THE MESSENGER-RNA POLY(A)-BINDING PROTEIN - LOCALIZATION, ABUNDANCE, AND RNA-BINDING SPECIFICITY

The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3'-po ly(A) tail found on most eukaryotic mRNAs and together with the poly(A ) tail has been implicated in governing the stability and the translat ion of mRNA. In order to further understand the role of the PABP in th ese processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the hum an PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hP ABP and confocal immunofluorescence microscopy with these antibodies r eveals that it is localized exclusively to the cytoplasm. The hPABP ex hibits a very low turnover rate in these cells and quantitative immuno blotting experiments demonstrated that growing HeLa cells contain a su rprisingly high number of approximately 8 X 10(6) PABP molecules per c ell, which corresponds to an intracellular concentration of about 4 mu M. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and i t binds oligo(rA)(25) with an apparent K-d of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (K-d gr eater than or equal to 0.5 mu M). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concent ration of the hPABP, which is three orders of magnitude above its K-d for oligo(rA)(25), suggest that the hPABP may bind to additional, lowe r affinity binding sites in vivo. (C) 1994 Academic Press, Inc.