Characterizing the pre-mRNA cleavage & polyadenylation site in C. elegans
During pre-mRNA processing, the Cleavage and Polyadenylation Complex (CPC) recognizes various elements in the 3' untranslated region (3'UTR). The CPC binds to the polyadenylation signal (PAS) before cleaving the pre-mRNA transcript and adding a string of adenosines (Poly(A) tail) to its polyadenylation site. Dysregulation of this process results in abnormal use of alternative polyadenylation sites within many transcripts, which has been linked to several cancers and other diseases. Previously, the Mangone lab found an enrichment of adenosines at the polyadenylation site in C. elegans (65% of transcripts). Additional in vivo experiments revealed that the CPC cleaves at nearby adenosines if the adenosine at the typical cleavage site is missing. However, the role these adenosines play in polyadenylation site selection remained poorly characterized. This study aimed to characterize how these terminal adenosines influence the locations of the polyadenylation site.
To further investigate this phenomenon, we generated 5 transgenic lines of C. elegans, which each contained a transgene with a terminal adenosine at various locations within its 3'UTR. RNA was extracted from several of these strains, converted to cDNA, and sequenced. The polyadenylation site shifted to upstream pyrimidines (cytosine or thymine) in sequenced mutants containing a terminal adenosine more than 16nt from the PAS, suggesting that there may be a limit to how far the polyadenylation site can be from the PAS before the CPC cleaves at pyrimidines upstream of the terminal adenosine. Further analysis indicated that the CPC may have a secondary preference to cleave after a string of pyrimidines, in addition to its known preference to cleave at an adenosine. Our findings will help further the scientific community's understanding of the CPC’s selection of polyadenylation sites, which could provide insight into the causes of dysregulation of alternative polyadenylation in disease.
