Nearly all eukaryotic pre-messenger RNAs must undergo cleavage and polyadenylation in their 3'-untranslated region (UTR) before being exported to the cytoplasm for translation into protein. The correct execution of these coupled and cotranscriptional processing events is critical for proper gene expression and normal cellular life. This topic has relevance to human health: certain heritable diseases cause illness through improper cleavage and polyadenylation. For example, the 1- and 2-thalassemias are caused by mutations in the processing signals of the 3'UTR of the globin genes. Approximately half of all human genes can be polyadenylated at different locations, a fact that can have regulatory consequences for an mRNA and its gene product, but little is known about the biochemical mechanisms by which such alternative polyadenylation takes place. We have previously found that a chromatography fraction containing both mammalian Cleavage Factors I and II (CFIm/CFIIm) loses activity when subjected to dephosphorylating enzymes in vitro. We propose that phosphorylation among these protein factors may play a role in their function and/or regulation. Our long-term goal is to understand how post-translational phosphorylation among the pre-mRNA 3'cleavage factors contributes to both the constitutive and regulated versions of this essential biochemical reaction.
Our specific aims for this work are: 1. To identify the human cleavage factor whose activity is determined by reversible phosphorylation? 2. To make and test recombinant CFIm heterodimers for phosphorylation-dependent in vitro reconstituted 3'cleavage, and cleavage rescue, activity. 3. To perform a mutational analysis of 3'cleavage activity dependence on CFIm phosphorylation. 4. To study pre-cleavage complex formation as a function of protein phosphorylation. Project Narrative This project will study the 3'cleavage and polyadenylation of messenger RNA. This topic has relevance to human health: certain heritable diseases cause illness through improper cleavage and polyadenylation. For example, the 1- and 2-thalassemias are caused by mutations in the processing signals of the 3'UTR of the globin genes.
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