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Venkataraman, Krishnan

Microbiology and Molecular Genetics

2005

Ph. D.

The process of synthesizing a mature mRNA transcript in eukaryotic cells is a multi-step event. The mRNA transcript is capped, spliced and polyadenylated before it can leave the nucleus for translation. The process of polyadenylation requires that the 3'-untranslated region (UTR) of the mRNA be recognized prior to the actual event. This event was primarily thought to occur in vertebrates via the recognition of primarily two elements. The first was the canonical hexamer AAUAAA and the second, a poorly defined U/GU rich downstream element (DSE). Different trans-acting factors bind to these two elements, namely the Cleavage and Polyadenylation Stimulation Factor (CPSF) that binds to the AAUAAA hexamer and the Cleavage stimulation Factor (CstF) that binds to the DSE. The dogma while good for a large number of genes, was inadequate in addressing the rather significant question of how genes with non-canonical hexamers and weak DSEs could be processes efficiently. Non-canonical hexamers and weak DSEs are known to be poor polyadenylation signals. It is far from trivial that these hexamer variants comprise more than 30% of all expressed genes. The role of an upstream sequence enhancer (USE) was shown to enhance some polyadenylation signals, but has largely remained undefined and its exact role, speculative at best. The role of the USE is significant in plants and in yeast (denoted as the positioning element) and is better defined, both in terms of cis and trans acting factors. In either case, the polyadenylation machinery appears to depend on all of the three sequence elements mentioned above. The dependence of apparently weak polyadenylation signals of vertebrate genes on additional sequences upstream of the hexamer locus and the DSE for efficient processing has been shown in this work. We show that the loss of CFI m binding in these USEs results in impaired polyadenylation. We have also successfully demonstrated that in the case of a weak hexamer, the binding of CFI m upstream is critical for 3'-processing in several UTRs. We have demonstrated that CFI m can physically and functionally interact with CPSF. We have also shown the recognition of a minimal 3' UTR recognition system using only rCFIm, rhFip 1 and PAPa on the UTR of PAPOLG. This work has allowed us to demonstrate a significant similarity to the organization of the polyadenylation signal between yeast, plants and vertebrates.