Differences in polyadenylation site choice between somatic and male germ cells.
BACKGROUND
We have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. First, male germ cells showed a lower incidence of the sequence AAUAAA (an important element for somatic polyadenylation site choice) near the polyadenylation site choice. Second, the polyadenylation sites chosen in male germ cells tended to be nearer the 5' end of the mRNA than those chosen in somatic cells. Finally, a number of mRNAs used a different polyadenylation site in male germ cells than in somatic cells. These differences suggested that male germ cell-specific polyadenylation sites may be poor substrates for polyadenylation in somatic cells. We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells.


RESULTS
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.


CONCLUSIONS
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.
McMahon
K Wyatt
KW
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6540 USA. kevin.mcmahon@ttuhsc.edu
Hirsch
Benjamin A
BA
MacDonald
Clinton C
CC
D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D009928:Organ Specificity; D026723:Polyadenylation / Q000235:genetics; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D013094:Spermatozoa / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D008565:Membrane Proteins; D012322:RNA Precursors; C096806:zonadhesin; D005914:Globins; D049410:Luciferases, Renilla