The cleavage and polyadenylation of pre-mRNAs is a critical step needed for RNA transcription termination and maturation. This process is executed by a large multi-subunit complex known as the RNA cleavage and polyadenylation complex (CPC). The CPC binds to the polyadenylation signal (PAS), which is a conserved hexameric element located at the end of the transcript's 3' Untranslated Region (3'UTR). The CPC then performs the cleavage reaction at the polyadenylation (PS) site. Despite their importance, PS element locations in eukaryotic genomes are poorly characterized. Prior research from our lab revealed that the distance between the PAS and the PS elements is not constant and a buffer region between 12-14 nt from the PAS element is needed by the CPC in order to perform a successful cleavage reaction. Our lab recently identified an enrichment of adenosine nucleotides at the PS site and demonstrated that their removal alters the location of the cleavage site in vivo, suggesting an important novel role of the PS element in pre-mRNA cleavage and polyadenylation. In order to further study the involvement of this terminal adenosine located at the PS site, we developed a novel in vivo cleavage and polyadenylation assay which we will use to determine the optimal buffer region length and further study the role of the terminal adenosine at the cleavage site. For these experiments, we have used the M03A1.3 3'UTR since it uses only one canonical PAS element (and thus does not use alternative polyadenylation) and has a buffer region of 14 nt. We have prepared a GFP reporter construct containing a mutated M03A1.3 3'UTR with no adenosines between the PAS and PS elements. This GFP reporter construct was used to prepare seven new mutants containing a terminal adenosine inserted at specific locations between 17 and 29 nucleotides downstream of the PAS site. The results of this assay will provide a working framework which we will use to model PS elements in the 5546 genes in the worm transcriptome, which currently lack annotated 3'UTR data. Our work will greatly improve our understanding of pre-mRNA cleavage and polyadenylation in C. elegans and will allow us to provide a needed reference for PS elements in the worm transcriptome to the scientific community.

Affiliations:
- School of Life Sciences, Arizona State University, Tempe, AZ
- The Biodesign Institute, Arizona State University, Tempe, AZ
- Molecular and Cellular Biology Graduate Program, Arizona State University, Tempe, AZ