Translational control of poly(A)-binding protein expression
Poly(A)-binding protein (PABP) is a highly conserved, ubiquitous, and essential polypeptide in eukaryotes. PABP, in conjugation with the 3'-poly(A)-tail, is shown to influence every aspects of mRNA metabolism including maturation, transportation, localization, stability and translation. Several studies indicate that PABP behaves like an early response gene and fluctuation in its cellular levels, in response to developmental or physiological conditions, is associated with dysregulation of the expression of several important genes. Given the significance of PABP in global gene regulation, controlling its cellular level is essential for normal cell functioning. The PABP-mRNA contains a highly conserved adenine-rich 'cis'-acting region, called autoregulatory sequence (ARS), in the 5'-UTR. Available evidence suggests that, at elevated cellular levels, PABP binds to the ARS and represses its own translation. However, the precise mechanism of the repression of PABP-mRNA translation by feedback inhibition is not known. This thesis presents an investigation of the mechanism of PABP autoregulation at the molecular level. Since all known functions of PABP are attributed to its ability to interact with the poly(A)-RNA tract and to act as a scaffold for protein-protein interactions, we hypothesize that PABP may recruits several other polypeptides to the ARS and form a large ribonucleoprotein complex that could stall the movement of 40S small ribosomal subunit at the 5'-UTR. We have shown that the ARS forms a complex 'in vitro' with PABP, and two additional polypeptides, IMP1, an ortholog of chicken zip-code binding polypeptide, and UNR, a PABP binding polypeptide. RNA-protein interaction studies show that at least three out of four RBD domains of PABP and KH III-IV domains of IMP1, respectively, are required for the interaction with the ARS RNA. In addition, PABP and IMP1 polypeptides also interact with each other through the PABP-C and KH III-IV domains, respectively. Mutational analyses show that the ability of ARS to form a PABP, IMP1 and UNR containing heteromeric ribonucleoprotein complex correlates well with its translational repressor activity. Results of our analysis suggest that the formation of this complex is required for maximal repression of PABP mRNA translation under physiological conditions. Our investigation in the translational control of PABP expression has raised interesting questions pertaining to the multifunctional roles of IMP1 and UNR polypeptides in gene regulation.