Differences in polyadenylation site choice between somatic and male germ cells

We tested whether pre-mRNA sequences surrounding male germ cell-specific polyadenylation sites (polyadenylation cassettes) could be used to direct polyadenylation efficiently in somatic cells. To do this, we developed a luciferase reporter system in which luciferase activity correlated with polyadenylation efficiency. We showed that in somatic cells, somatic polyadenylation cassettes were efficiently polyadenylated, while male germ cell-specific polyadenylation cassettes were not. We also developed a sensitive, 3' RACE-based assay to analyze polyadenylation site choice. Using this assay, we demonstrated that male germ cell-specific polyadenylation cassettes were not polyadenylated at the expected site in somatic cells, but rather at aberrant sites upstream of the sites used in male germ cells. Finally, mutation of the male germ cell-specific poly(A) signal to a somatic poly(A) signal resulted in more efficient polyadenylation in somatic cells.These data suggest that regulated polyadenylation site choice of male germ cell-specific polyadenylation sites requires one or more factors that are absent from somatic cells.Polyadenylation occurs in three stages: polyadenylation site choice, cleavage of the pre-mRNA, and addition of the poly(A) tail to the newly formed 3' end [1,2]. The first step, polyadenylation site choice, can be defined as the functional assembly of the factors necessary for pre-mRNA cleavage onto the pre-mRNA to allow for efficient, accurate cleavage of the pre-mRNA (it has also been called the commitment step) [3-5]. Mutation of the pre-mRNA sequence elements involved in polyadenylation site choice [6-12], or mutation of the protein machinery involved in polyadenylation site choice [13-15], result in inefficient polyadenylation of the pre-mRNA. Consequently, inefficient polyadenylation prevents export of mRNA and decreases production of the protein encoded by that mRNA [1]. Therefore, polyadenylation site choice is an important first step in polyadenyl