Post-transcriptional control of mRNA stability is a major mechanism by which many hormones regulate gene expression. Estrogen cause the translational reprogramming of Xenopus hepatocytes by inducing both the destabilization of serum protein mRNAs and the transcriptional induction and stabilization of yolk protein mRNA. The loss of serum protein mRNAs results from the selective activation of the polysome-associated mRNA endonuclease PMR-1. PMR-1 exists in a latent form on polysomes as part of a greater than 670 kDa mRNP complex, and it is in the context of this complex that mRNA decay is affected by a hormone-induced 22- fold increase in unit enzymatic activity. PMR-1 is the first vertebrate mRNA endonuclease to be characterized, and understanding the assembly of PMR-1 into the mRNP complex and the process of its hormonal activation will provide key insights into a major pathway of gene expression and its regulation. The current proposal will focus on characterizing the assembly of PMR-1 into the greater than 670 kDa mRNP complex and the role of the estrogen receptor in the activation of PMR-1 and mRNA decay.
The Specific Aims for this project are to: 1) determine the domains of PMR-1 involved in targeting the mRNA endonuclease to the greater than 670 kDa complex using cells transfected with vectors expressing recombinant protein; 2) identify proteins that interact with PMR-1 using expression screening of cDNA libraries, co-precipitation, and a novel affinity purification scheme for selective recovery of mRNP complexes from cells; 3) characterize the interactions both in vitro and in vivo between PMR-1 and the PMR-1-interacting proteins identified in Aim 2 using co- precipitation and functional assays for effects on mRNA decay, and map interacting domains between these proteins; and 4) evaluate the role of estrogen receptor in the activation of mRNA decay, determine receptor domains required for this process, and characterize the effects of estrogen on the activation of PMR-1 and its association with proteins in the mRNP complex.
Showing the most recent 10 out of 58 publications
