mRNA Poly(A) tail: a 3' Enhancer of Translational Initiation: a Thesis

Most eukaryotic mRNAs have a sequence of polyadenylic acid [poly(A)] at their 3'-termini. Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) have yet to be clarified. Earlier results from our laboratory led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of poly(A) + , but not poly(A) - , mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include: 1) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency in vivo and in vitro; 2) correlations between the abundance and stability of PABPs and the rate of translational initiation in vivo and in vitro; and 3) the demonstration that exogenous poly(A) is a potent and specific inhibitor of the in vitro translation of poly(A) + , but not poly(A) - mRNAs. To evaluate the hypothesis that the 3'-poly(A) tract of mRNA plays a role in translational initiation, we have constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs are translated, degraded, recruited into polysomes, and assembled into mRNPs or intermediates in the translational initiation pathway. Relative to mRNAs which are polyadenylated, we find that poly(A) - mRNAs have a reduced translational capacity which is not due to an increase in their decay rates, but is attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A) - mRNAs affects a late step in translational initiation, is distinct from the phenotype associated with cap-deficient mRNAs, and results in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibits translation from capped poly(A) + mRNAs, but stimulates translation from capped poly(A) - mRNAs. We suggest that poly(A) is the formal equivalent of a transcriptional enhancer, i.e., that poly(A)-binding protein (PABP) bound at the 3'-end of mRNA may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5'-end.