The long-term goal of this project is to understand the molecular mechanisms of eukaryotic mRNA polyadenylation. mRNA polyadenylation plays an essential role in the initiation step of protein synthesis, in the export of mRNAs from the nucleus to the cytoplasm, and in the control of mRNA stability. Polyadenylation is a key regulatory step in the expression of many genes. Aberrant polyadenylation has been shown to cause diseases such as thalassemia and lysosomal storage disorder. Moreover, oculopharyngeal muscular dystrophy is the result of the insertion of short GCG repeats in the gene encoding one of the polyadenylation factors, poly(A) binding protein 2 (PABP 2). We are investigating the crystal structure of the enzyme at the heart of the polyadenylation machinery, poly(A) polymerase (PAP), its interaction with substrates, and its association with proteins playing a part in mRNA 3'-end processing. There are no structural data to date for any of the mammalian polyadenylation factors.
The specific aims are as follows: 1. The X-ray crystal structure of bovine PAP with its substrates ATP and poly(A) RNA will be determined using a combination of multiwavelength anomalous diffraction (MAD) and multiple isomorphous replacement. The structure of PAP complexed with substrates will guide additional structural and functional studies. 2. PABP 2 is required for processive synthesis and control of the poly(A) tail length. PABP2 is known to bind both the poly(A) tail and PAP. We will work towards the structure determination of the ternary complex of PABP2, PAP, and poly(A), using either the intact proteins or the interacting domains of each protein. 3. Phosphorylation of target sites located in the C-terminal domain of PAP results in strong repression of PAP activity. The down regulation of PAP via hyperphosphorylation is reminiscent of the inhibitory effect of U1A, which has been shown to inhibit polyadenylation of its own mRNA by binding to PAP. We will work towards the crystallization of the complex between PAP and U1A, using either the intact proteins, or the C-termini of each protein. We will concurrently attempt to crystallize phosphorylated, full-length bovine PAP. A comparison of the phosphorylated PAP structure with that of the PAP-U1A complex should elucidate whether both situations use a similar mechanism of repression. It is expected that these results will not only provide a sound structural basis for understanding the mechanism of polyadenylation at the molecular level but will also shed light on the mechanisms of processivity and repression.
