BACKGROUND
polyadenylation occurs in three stages: polyadenylation site choice, cleavage of the pre-mrna, and addition of the poly tail to the newly formed 3' end  <cit> .
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   <cit> .
mutation of the pre-mrna sequence elements involved in polyadenylation site choice  <cit> , or mutation of the protein machinery involved in polyadenylation site choice  <cit> , 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  <cit> .
therefore, polyadenylation site choice is an important first step in polyadenylation and is essential for optimal gene expression.
in mammalian somatic cells, the mechanism of polyadenylation site choice has been intensely studied  <cit> .
a number of pre-mrna sequences have been proposed to be important in choosing the site of polyadenylation  <cit> ; however, two seem to play a prominent role in mammalian somatic cells.
the first is the hexameric poly signal  found 15– <dig> bases upstream of the site of polyadenylation  <cit> .
the other is the g/u-rich element found 20– <dig> bases downstream of the site of polyadenylation  <cit> .
together, these elements bind the multi-subunit cleavage and polyadenylation specificity factor   <cit>  and cleavage stimulation factor   <cit> , respectively.
thus, the formation of this protein/rna complex determines the polyadenylation site choice.
the next step is cleavage of the pre-mrna at the polyadenylation site  followed by addition of the poly tail  <cit> .
however, a number of mrnas use different polyadenylation sites in different tissues or developmental stages  <cit> .
changes in the composition of the protein polyadenylation machinery can invoke a change in polyadenylation site choice called alternative polyadenylation  <cit> .
in addition, inclusion or exclusion of pre-mrna sequences outside of the polyadenylation region  may also affect polyadenylation site choice  <cit> .
therefore, changes in the protein/pre-mrna complex involved in polyadenylation site choice can change where the poly tail is added, and thereby affect gene expression.
we have noticed that the polyadenylation sites chosen in male germ cells are different from those chosen in somatic cells  <cit> .
first, a number of mrnas use a polyadenylation site at higher frequency than in other tissues  <cit> .
second, the incidence of the sequence aauaaa near the 3' ends of male germ cell mrnas is lower than in somatic mrnas  <cit> .
third, the polyadenylation sites chosen in male germ cell mrnas often result in shorter 3' untranslated regions than somatic mrnas  <cit> .
this suggests that there are significant differences in the polyadenylation sites chosen in somatic and male germ cells.
there are two possible causes of these differences.
either male germ cell-enriched polyadenylation sites can be used in somatic cells but are not , or they are poor substrates for polyadenylation in somatic cells.
we hypothesized that male germ cell-specific polyadenylation sites are poor substrates for polyadenylation in somatic cells, and therefore would be used inefficiently in somatic cells.
to test this, we developed a luciferase-based reporter assay to evaluate the polyadenylation efficiency of different sequences.
we then used the assay to show that sequences surrounding male germ cell-specific polyadenylation sites  were inefficiently polyadenylated in somatic cells.
additionally, we developed a 3' race-based approach to analyze polyadenylation site positioning.
using this approach, we observed that mrnas containing these male germ cell-specific polyadenylation sites were not polyadenylated at the site chosen in male germ cells.
rather, they showed aberrant polyadenylation upstream of the male germ cell-specific polyadenylation site.
finally, we showed that introduction of an aauaaa  into a male germ cell-specific pre-mrna allowed for more efficient polyadenylation of that site in somatic cells.
these data suggested that male germ cell-specific polyadenylation sites were inefficiently chosen in somatic cells, and that polyadenylation site choice has different requirements in male germ cells than in somatic cells.
RESULTS
development of a luciferase reporter system to assay polyadenylation efficiency
to test whether male germ cell-specific polyadenylation sites were inefficiently polyadenylated in somatic cells, we developed a system to assay polyadenylation efficiency.
we used pcr to isolate genomic sequences surrounding various polyadenylation sites used in vivo; we called these sequences polyadenylation cassettes .
each polyadenylation cassette was between  <dig> and  <dig> base pairs long, approximately centered on the site of polyadenylation.
each polyadenylation cassette was separately sub-cloned downstream of the coding region of renilla luciferase  to make a reporter plasmid.
this reporter plasmid was then co-transfected into somatic cells  with a separate plasmid expressing firefly luciferase to control for transfection efficiency .
since efficient polyadenylation is necessary for gene expression  <cit> , relative luciferase activity  was used as a measure of polyadenylation efficiency for each polyadenylation cassette.
similar assay systems have been developed using the cat reporter system  <cit> .
to determine whether our assay measures polyadenylation efficiency, we tested three previously characterized polyadenylation cassettes.
the first two, the sv <dig> late and the rabbit β-globin polyadenylation cassettes, are strong polyadenylation cassettes, and were therefore used as a reference for efficient polyadenylation.
the third was a sv <dig> late mutant cassette, which was the result of mutating the poly signal from aauaaa to gagaaa.
mutating the sv <dig> aauaaa was previously shown to prevent binding of the polyadenylation machinery to the pre-mrna, thus preventing polyadenylation site choice and resulting in inefficient polyadenylation  <cit> .
we transfected mouse embryonic fibroblast cells  with the above reporter plasmids and  <dig> hours later lysed the cells and assayed thy lysates for luciferase activity.
pilot experiments were performed to determine conditions for transfection that yielded a linear increase in renilla luciferase activity .
as expected, lysates from cells transfected with reporter plasmids containing the wild type sv <dig> late or rabbit β-globin polyadenylation cassettes showed significantly higher levels of luciferase than those lysates from cells transfected with the mutant-containing reporter plasmid .
interestingly, lysates from cells transfected with reporter plasmids containing the sv <dig> late polyadenylation cassette showed  <dig>  times higher levels of luciferase activity than those containing the rabbit β-globin cassette .
this could be because the sv <dig> late polyadenylation cassette is viral, and therefore highly efficient in many tissues, whereas the rabbit β-globin cassette is being expressed in mouse fibroblasts rather than in rabbit reticulocytes .
these data demonstrate that the luciferase activity in lysates from these cells correlates with polyadenylation efficiency.
to test whether a male germ cell-specific polyadenylation cassette could be efficiently polyadenylated in somatic cells, another reporter plasmid was created with the polyadenylation cassette from the zonadhesin pre-mrna.
since the zonadhesin mrna is expressed only in male germ cells  <cit> , it was used as a reference for male germ cell-specific polyadenylation.
similar to the results using the sv <dig> mutant reporter plasmid, the level of luciferase activity in lysates from cells transfected with the reporter plasmid containing the zonadhesin cassette was significantly lower than the level in lysates from cells transfected with the sv40-containing reporter plasmid .
therefore, the luciferase activity present in lysates of cells transfected with a reporter plasmid containing a male germ cell-specific polyadenylation cassette was similar to the level found in lysates from cells transfected with an inefficiently polyadenylated cassette.
genes with male germ cell-specific polyadenylation sites are expressed at lower levels than those with somatic polyadenylation sites from the same mrna
the sv <dig> wild type and rabbit β-globin polyadenylation cassettes are well-characterized  <cit> .
in order to determine whether male germ cell-specific polyadenylation sites are truly inefficiently polyadenylated relative to somatic polyadenylation sites in somatic cells, we compared the polyadenylation efficiency of somatic and male germ cell-specific cassettes that both derive from the same gene.
since cyclin a <dig> potentially can use two different polyadenylation sites in somatic cells and two different polyadenylation sites in male germ cells  <cit> , mouse embryonic fibroblasts were transfected with reporter plasmids containing either the somatic or the male germ cell-specific polyadenylation cassettes from the cyclin a <dig> mrna.
luciferase activity from lysates of these transfected cells was then assayed.
as shown in figure 2a, lysates from cells transfected with the reporter plasmids containing the somatic- <dig> and somatic- <dig> polyadenylation cassettes from cyclin a <dig> showed much higher luciferase activity than did lysates from cells transfected with male germ cell-specific- <dig> and male germ cell-specific- <dig> polyadenylation cassette-containing plasmids.
therefore, for the cyclin a <dig> pre-mrna, transfection of cells with reporter plasmids containing the somatic polyadenylation cassettes resulted in expression of higher levels of luciferase activity than those containing the male germ cell-specific polyadenylation cassettes.
these studies were extended to compare the polyadenylation efficiencies of the various crem and c-abl polyadenylation cassettes, each of which uses one polyadenylation site in somatic cells and a different site in male germ cells  <cit> .
we transfected fibroblasts with reporter plasmids containing polyadenylation cassettes from c-abl and crem mrnas.
again, lysates from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes showed lower levels of luciferase activity than did those lysates with somatic cassettes in the reporter plasmids .
thus, even when studied independently of each other, the somatic polyadenylation sites are associated with higher levels of luciferase activities in extracts from transfected cells.
male germ cell-specific polyadenylation sites are inefficiently used in somatic cells
we wanted to determine whether the low levels of luciferase activity we observed in extracts from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes were because of problems with polyadenylation site choice.
to do this, we transfected mouse embryonic fibroblasts with reporter plasmids used in earlier experiments, extracted rna from the transfected cells, made cdna, and subjected it to 3' race.
the products were purified, cloned, and sequenced, and the sequences were aligned with the reporter plasmid with which these cells were transfected in order to determine the site of polyadenylation .
in contrast, all five cdnas cloned from cells transfected with the sv <dig> late mutant polyadenylation cassette-containing reporter plasmid showed polyadenylation at aberrant, upstream positions, and never at the reported site of polyadenylation .
this upstream polyadenylation is different from the read through transcripts most investigators have observed because of inefficient polyadenylation  <cit> .
in addition, the cdnas cloned from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes all showed aberrant upstream polyadenylation, similar to that seen with the sv <dig> mutant.
interestingly, most of the aberrant polyadenylation sites were found in the luciferase coding region.
therefore, all of the polyadenylation cassettes associated with low levels of luciferase give rise to aberrantly polyadenylated mrnas when transfected into somatic cells.
sequences in cassettes affect polyadenylation efficiency
cis-acting sequences around the site of polyadenylation have been shown to affect polyadenylation efficiency profoundly  <cit> .
we hypothesized that the reason male germ cell-specific polyadenylation sites were not chosen in somatic cells was because they lacked the necessary cis-acting sequences that somatic polyadenylation site choice requires.
if this were true, altering the sequences on a male germ cell-specific polyadenylation cassette would increase the ability of a male germ cell-specific polyadenylation site to be chosen in somatic cells.
to test this hypothesis, we created four polyadenylation cassettes.
three of them  were used in earlier studies.
we also created a zonadhesin mutant, which converted a putative male germ cell-specific poly signal gagaaa to an aauaaa.
cells were transfected with reporter plasmids containing each of these four polyadenylation cassettes and subsequently lysates were assayed for luciferase activity.
as before, cells transfected with the sv <dig> late-containing reporter plasmid showed high levels of relative luciferase activity, and lysates from cells transfected with sv <dig> late mutant and zonadhesin-containing reporter plasmids showed significantly lower levels of relative activity .
however, when cells were transfected with the reporter plasmids containing a zonadhesin mutant polyadenylation cassette, significantly higher levels of luciferase activity were observed when compared to the zonadhesin-containing reporter plasmid.
therefore, altering the poly signal in a male germ cell-specific polyadenylation cassette can increase the luciferase activity in extracts from cells transfected with this plasmid.
discussion and
CONCLUSIONS
our observations  <cit> , as well as studies done by others  <cit> , suggested that there were differences in how somatic and male germ cells choose a polyadenylation site.
however, we had never tested directly whether somatic cells were capable of efficiently choosing a polyadenylation site from a male germ cell-specific mrna.
we therefore devised a luciferase reporter assay that measures polyadenylation efficiency.
using this assay, we showed that extracts from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes showed significantly lower levels of luciferase activity than extracts from cells transfected with somatic cassette-containing plasmids.
extracts of cells transfected with reporter plasmids containing somatic polyadenylation cassettes also expressed higher levels of luciferase activity than those with male germ cell-specific polyadenylation cassettes from the same mrna.
analysis of 3' race products showed that mrnas containing somatic polyadenylation cassettes were polyadenylated almost exclusively at the reported sites.
mutation of the aauaaa polyadenylation signal to a gagaaa resulted in no observed polyadenylation at the reported sites.
rather, analysis of the 3' ends of these mrnas showed that any mrnas that were being polyadenylated were being polyadenylated at aberrant sites.
finally, introduction of an aauaaa into a male germ cell-specific polyadenylation cassette resulted in significantly higher levels of luciferase in extracts transfected with this plasmid.
these data suggest that male germ cell-specific polyadenylation sites are inefficiently used in somatic cells.
each of the male germ cell-specific polyadenylation sites shown here are efficiently used in male germ cells  <cit> .
additionally, we have shown that the somatic polyadenylation sites for these pre-mrnas are used in somatic cells .
why, then, are male germ cell-specific polyadenylation sites inefficiently used in somatic cells?
as discussed above, polyadenylation site choice is the product of the sequences present on the pre-mrna and the polyadenylation machinery present in the cell.
there is a lower incidence of the sequence aauaaa near the 3' ends of mrnas in male germ cells relative to the incidence in other tissues  <cit> .
other studies have suggested that other elements essential for polyadenylation in somatic cells may likewise be absent from male germ cell mrnas  <cit> .
the fact that male germ cell-specific polyadenylation cassettes often lack the sequences necessary for somatic polyadenylation could be the reason for male germ cell-specific polyadenylation sites not being chosen in somatic cells.
additionally, it is possible that elements in male germ cell-specific polyadenylation cassettes prevent their polyadenylation in somatic cells.
however, this study has shown that male germ cell-specific polyadenylation sites are used rarely, if at all in typical somatic cells .
introduction of a somatic polyadenylation signal  can greatly increase its ability to be chosen in these cells , but there are likely other elements involved as well.
this suggests that the polyadenylation machinery present in somatic cells is less compatible with the polyadenylation sequences present in male germ cell-specific mrnas than with somatically expressed mrnas.
additionally, male germ cells express proteins that are present either exclusively or predominantly in this tissue that are homologous to known polyadenylation proteins  <cit> .
two of these proteins, a cstf- <dig> homolog called wdc <dig>  <cit>  and a cstf- <dig> homolog called τcstf- <dig>  <cit> , are similar to proteins that have been associated with changing polyadenylation site choice  <cit> .
the presence of these proteins in male germ cells may alter the polyadenylation machinery's preference for polyadenylation site choice, and their absence may explain why male germ cell-specific polyadenylation sites are inefficiently chosen in somatic cells.
we originally hypothesized that τcstf- <dig> would affect polyadenylation site choice for the mrnas studied here  <cit> .
however, we have recently shown that the crem, c-abl, and ccna <dig> mrnas are expressed at normal levels and are polyadenylated at the same position in the testis of mice lacking τcstf- <dig> as in the wild type testis .
this strongly suggests that τcstf- <dig> is not involved in changing the polyadenylation site choice for these transcripts.
however, other proteins expressed in male germ cells – proteins that are also absent from fibroblasts – could be responsible for the change in polyadenylation site choice for these male germ cell-specific mrnas.
one surprising finding was of the existence of aberrantly polyadenylated mrnas with poly site choice occurring upstream of the reported site of polyadenylation .
previous studies showed that if a pre-mrna was inefficiently polyadenylated, then rna polymerase ii read through the site of polyadenylation, producing longer transcripts  <cit> .
in contrast, we observed mrnas that were polyadenylated at disparate places, all upstream of the reported site and several in the luciferase coding region.
we used a 3' race-based approach to look for changes in polyadenylation; this is opposed to the rnase protection assays and nuclear run-on assays previous investigators have used  <cit> .
because we saw this aberrant polyadenylation associated with the sv <dig> late mutant polyadenylation cassette, we believe that aberrant polyadenylation is the result of inefficient polyadenylation.
it is also possible that these aberrant polyadenylation sites often occur naturally, but are only detectable in the absence of efficient polyadenylation.
however, we propose that the use of a more sensitive pcr-based method has allowed us to observe previously undescribed upstream aberrant polyadenylation.
finally, our data suggest the possibility that male germ cells express one or more different factors and have different sequence requirements that together alter how the polyadenylation site is chosen.
other tissues may similarly have different mechanisms of polyadenylation site choice.
the brain seems especially prone to contain mrnas that are alternatively polyadenylated  <cit> , and our lab has identified a brain-specific form of cstf- <dig> , which may alter polyadenylation site choice in this tissue.
the existence of such alternative methods of polyadenylation site choice could be used to greatly increase the protein diversity within a cell.

 polyadenylation occurs in three stages: polyadenylation site choice, cleavage of the pre-mrna, and addition of the poly tail to the newly formed 3' end. 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. mutation of the pre-mrna sequence elements involved in polyadenylation site choice, or mutation of the protein machinery involved in polyadenylation site choice, 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. therefore, polyadenylation site choice is an important first step in polyadenylation and is essential for optimal gene expression. in mammalian somatic cells, the mechanism of polyadenylation site choice has been intensely studied. a number of pre-mrna sequences have been proposed to be important in choosing the site of polyadenylation; however, two seem to play a prominent role in mammalian somatic cells. the first is the hexameric poly signal found 15– bases upstream of the site of polyadenylation. the other is the g/u-rich element found 20– bases downstream of the site of polyadenylation. together, these elements bind the multi-subunit cleavage and polyadenylation specificity factor and cleavage stimulation factor, respectively. thus, the formation of this protein/rna complex determines the polyadenylation site choice. the next step is cleavage of the pre-mrna at the polyadenylation site followed by addition of the poly tail. however, a number of mrnas use different polyadenylation sites in different tissues or developmental stages. changes in the composition of the protein polyadenylation machinery can invoke a change in polyadenylation site choice called alternative polyadenylation. in addition, inclusion or exclusion of pre-mrna sequences outside of the polyadenylation region may also affect polyadenylation site choice. therefore, changes in the protein/pre-mrna complex involved in polyadenylation site choice can change where the poly tail is added, and thereby affect gene expression. we have noticed that the polyadenylation sites chosen in male germ cells are different from those chosen in somatic cells. first, a number of mrnas use a polyadenylation site at higher frequency than in other tissues. second, the incidence of the sequence aauaaa near the 3' ends of male germ cell mrnas is lower than in somatic mrnas. third, the polyadenylation sites chosen in male germ cell mrnas often result in shorter 3' untranslated regions than somatic mrnas. this suggests that there are significant differences in the polyadenylation sites chosen in somatic and male germ cells. there are two possible causes of these differences. either male germ cell-enriched polyadenylation sites can be used in somatic cells but are not, or they are poor substrates for polyadenylation in somatic cells. we hypothesized that male germ cell-specific polyadenylation sites are poor substrates for polyadenylation in somatic cells, and therefore would be used inefficiently in somatic cells. to test this, we developed a luciferase-based reporter assay to evaluate the polyadenylation efficiency of different sequences. we then used the assay to show that sequences surrounding male germ cell-specific polyadenylation sites were inefficiently polyadenylated in somatic cells. additionally, we developed a 3' race-based approach to analyze polyadenylation site positioning. using this approach, we observed that mrnas containing these male germ cell-specific polyadenylation sites were not polyadenylated at the site chosen in male germ cells. rather, they showed aberrant polyadenylation upstream of the male germ cell-specific polyadenylation site. finally, we showed that introduction of an aauaaa into a male germ cell-specific pre-mrna allowed for more efficient polyadenylation of that site in somatic cells. these data suggested that male germ cell-specific polyadenylation sites were inefficiently chosen in somatic cells, and that polyadenylation site choice has different requirements in male germ cells than in somatic cells.  development of a luciferase reporter system to assay polyadenylation efficiency to test whether male germ cell-specific polyadenylation sites were inefficiently polyadenylated in somatic cells, we developed a system to assay polyadenylation efficiency. we used pcr to isolate genomic sequences surrounding various polyadenylation sites used in vivo; we called these sequences polyadenylation cassettes. each polyadenylation cassette was between and base pairs long, approximately centered on the site of polyadenylation. each polyadenylation cassette was separately sub-cloned downstream of the coding region of renilla luciferase to make a reporter plasmid. this reporter plasmid was then co-transfected into somatic cells with a separate plasmid expressing firefly luciferase to control for transfection efficiency. since efficient polyadenylation is necessary for gene expression, relative luciferase activity was used as a measure of polyadenylation efficiency for each polyadenylation cassette. similar assay systems have been developed using the cat reporter system. to determine whether our assay measures polyadenylation efficiency, we tested three previously characterized polyadenylation cassettes. the first two, the sv late and the rabbit β-globin polyadenylation cassettes, are strong polyadenylation cassettes, and were therefore used as a reference for efficient polyadenylation. the third was a sv late mutant cassette, which was the result of mutating the poly signal from aauaaa to gagaaa. mutating the sv aauaaa was previously shown to prevent binding of the polyadenylation machinery to the pre-mrna, thus preventing polyadenylation site choice and resulting in inefficient polyadenylation. we transfected mouse embryonic fibroblast cells with the above reporter plasmids and hours later lysed the cells and assayed thy lysates for luciferase activity. pilot experiments were performed to determine conditions for transfection that yielded a linear increase in renilla luciferase activity. as expected, lysates from cells transfected with reporter plasmids containing the wild type sv late or rabbit β-globin polyadenylation cassettes showed significantly higher levels of luciferase than those lysates from cells transfected with the mutant-containing reporter plasmid. interestingly, lysates from cells transfected with reporter plasmids containing the sv late polyadenylation cassette showed times higher levels of luciferase activity than those containing the rabbit β-globin cassette. this could be because the sv late polyadenylation cassette is viral, and therefore highly efficient in many tissues, whereas the rabbit β-globin cassette is being expressed in mouse fibroblasts rather than in rabbit reticulocytes. these data demonstrate that the luciferase activity in lysates from these cells correlates with polyadenylation efficiency. to test whether a male germ cell-specific polyadenylation cassette could be efficiently polyadenylated in somatic cells, another reporter plasmid was created with the polyadenylation cassette from the zonadhesin pre-mrna. since the zonadhesin mrna is expressed only in male germ cells, it was used as a reference for male germ cell-specific polyadenylation. similar to the results using the sv mutant reporter plasmid, the level of luciferase activity in lysates from cells transfected with the reporter plasmid containing the zonadhesin cassette was significantly lower than the level in lysates from cells transfected with the sv40-containing reporter plasmid. therefore, the luciferase activity present in lysates of cells transfected with a reporter plasmid containing a male germ cell-specific polyadenylation cassette was similar to the level found in lysates from cells transfected with an inefficiently polyadenylated cassette. genes with male germ cell-specific polyadenylation sites are expressed at lower levels than those with somatic polyadenylation sites from the same mrna the sv wild type and rabbit β-globin polyadenylation cassettes are well-characterized. in order to determine whether male germ cell-specific polyadenylation sites are truly inefficiently polyadenylated relative to somatic polyadenylation sites in somatic cells, we compared the polyadenylation efficiency of somatic and male germ cell-specific cassettes that both derive from the same gene. since cyclin a potentially can use two different polyadenylation sites in somatic cells and two different polyadenylation sites in male germ cells, mouse embryonic fibroblasts were transfected with reporter plasmids containing either the somatic or the male germ cell-specific polyadenylation cassettes from the cyclin a mrna. luciferase activity from lysates of these transfected cells was then assayed. as shown in figure 2a, lysates from cells transfected with the reporter plasmids containing the somatic- and somatic- polyadenylation cassettes from cyclin a showed much higher luciferase activity than did lysates from cells transfected with male germ cell-specific- and male germ cell-specific- polyadenylation cassette-containing plasmids. therefore, for the cyclin a pre-mrna, transfection of cells with reporter plasmids containing the somatic polyadenylation cassettes resulted in expression of higher levels of luciferase activity than those containing the male germ cell-specific polyadenylation cassettes. these studies were extended to compare the polyadenylation efficiencies of the various crem and c-abl polyadenylation cassettes, each of which uses one polyadenylation site in somatic cells and a different site in male germ cells. we transfected fibroblasts with reporter plasmids containing polyadenylation cassettes from c-abl and crem mrnas. again, lysates from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes showed lower levels of luciferase activity than did those lysates with somatic cassettes in the reporter plasmids. thus, even when studied independently of each other, the somatic polyadenylation sites are associated with higher levels of luciferase activities in extracts from transfected cells. male germ cell-specific polyadenylation sites are inefficiently used in somatic cells we wanted to determine whether the low levels of luciferase activity we observed in extracts from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes were because of problems with polyadenylation site choice. to do this, we transfected mouse embryonic fibroblasts with reporter plasmids used in earlier experiments, extracted rna from the transfected cells, made cdna, and subjected it to 3' race. the products were purified, cloned, and sequenced, and the sequences were aligned with the reporter plasmid with which these cells were transfected in order to determine the site of polyadenylation. in contrast, all five cdnas cloned from cells transfected with the sv late mutant polyadenylation cassette-containing reporter plasmid showed polyadenylation at aberrant, upstream positions, and never at the reported site of polyadenylation. this upstream polyadenylation is different from the read through transcripts most investigators have observed because of inefficient polyadenylation. in addition, the cdnas cloned from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes all showed aberrant upstream polyadenylation, similar to that seen with the sv mutant. interestingly, most of the aberrant polyadenylation sites were found in the luciferase coding region. therefore, all of the polyadenylation cassettes associated with low levels of luciferase give rise to aberrantly polyadenylated mrnas when transfected into somatic cells. sequences in cassettes affect polyadenylation efficiency cis-acting sequences around the site of polyadenylation have been shown to affect polyadenylation efficiency profoundly. we hypothesized that the reason male germ cell-specific polyadenylation sites were not chosen in somatic cells was because they lacked the necessary cis-acting sequences that somatic polyadenylation site choice requires. if this were true, altering the sequences on a male germ cell-specific polyadenylation cassette would increase the ability of a male germ cell-specific polyadenylation site to be chosen in somatic cells. to test this hypothesis, we created four polyadenylation cassettes. three of them were used in earlier studies. we also created a zonadhesin mutant, which converted a putative male germ cell-specific poly signal gagaaa to an aauaaa. cells were transfected with reporter plasmids containing each of these four polyadenylation cassettes and subsequently lysates were assayed for luciferase activity. as before, cells transfected with the sv late-containing reporter plasmid showed high levels of relative luciferase activity, and lysates from cells transfected with sv late mutant and zonadhesin-containing reporter plasmids showed significantly lower levels of relative activity. however, when cells were transfected with the reporter plasmids containing a zonadhesin mutant polyadenylation cassette, significantly higher levels of luciferase activity were observed when compared to the zonadhesin-containing reporter plasmid. therefore, altering the poly signal in a male germ cell-specific polyadenylation cassette can increase the luciferase activity in extracts from cells transfected with this plasmid. discussion and  our observations, as well as studies done by others, suggested that there were differences in how somatic and male germ cells choose a polyadenylation site. however, we had never tested directly whether somatic cells were capable of efficiently choosing a polyadenylation site from a male germ cell-specific mrna. we therefore devised a luciferase reporter assay that measures polyadenylation efficiency. using this assay, we showed that extracts from cells transfected with reporter plasmids containing male germ cell-specific polyadenylation cassettes showed significantly lower levels of luciferase activity than extracts from cells transfected with somatic cassette-containing plasmids. extracts of cells transfected with reporter plasmids containing somatic polyadenylation cassettes also expressed higher levels of luciferase activity than those with male germ cell-specific polyadenylation cassettes from the same mrna. analysis of 3' race products showed that mrnas containing somatic polyadenylation cassettes were polyadenylated almost exclusively at the reported sites. mutation of the aauaaa polyadenylation signal to a gagaaa resulted in no observed polyadenylation at the reported sites. rather, analysis of the 3' ends of these mrnas showed that any mrnas that were being polyadenylated were being polyadenylated at aberrant sites. finally, introduction of an aauaaa into a male germ cell-specific polyadenylation cassette resulted in significantly higher levels of luciferase in extracts transfected with this plasmid. these data suggest that male germ cell-specific polyadenylation sites are inefficiently used in somatic cells. each of the male germ cell-specific polyadenylation sites shown here are efficiently used in male germ cells. additionally, we have shown that the somatic polyadenylation sites for these pre-mrnas are used in somatic cells. why, then, are male germ cell-specific polyadenylation sites inefficiently used in somatic cells? as discussed above, polyadenylation site choice is the product of the sequences present on the pre-mrna and the polyadenylation machinery present in the cell. there is a lower incidence of the sequence aauaaa near the 3' ends of mrnas in male germ cells relative to the incidence in other tissues. other studies have suggested that other elements essential for polyadenylation in somatic cells may likewise be absent from male germ cell mrnas. the fact that male germ cell-specific polyadenylation cassettes often lack the sequences necessary for somatic polyadenylation could be the reason for male germ cell-specific polyadenylation sites not being chosen in somatic cells. additionally, it is possible that elements in male germ cell-specific polyadenylation cassettes prevent their polyadenylation in somatic cells. however, this study has shown that male germ cell-specific polyadenylation sites are used rarely, if at all in typical somatic cells. introduction of a somatic polyadenylation signal can greatly increase its ability to be chosen in these cells, but there are likely other elements involved as well. this suggests that the polyadenylation machinery present in somatic cells is less compatible with the polyadenylation sequences present in male germ cell-specific mrnas than with somatically expressed mrnas. additionally, male germ cells express proteins that are present either exclusively or predominantly in this tissue that are homologous to known polyadenylation proteins. two of these proteins, a cstf- homolog called wdc and a cstf- homolog called τcstf-, are similar to proteins that have been associated with changing polyadenylation site choice. the presence of these proteins in male germ cells may alter the polyadenylation machinery's preference for polyadenylation site choice, and their absence may explain why male germ cell-specific polyadenylation sites are inefficiently chosen in somatic cells. we originally hypothesized that τcstf- would affect polyadenylation site choice for the mrnas studied here. however, we have recently shown that the crem, c-abl, and ccna mrnas are expressed at normal levels and are polyadenylated at the same position in the testis of mice lacking τcstf- as in the wild type testis. this strongly suggests that τcstf- is not involved in changing the polyadenylation site choice for these transcripts. however, other proteins expressed in male germ cells – proteins that are also absent from fibroblasts – could be responsible for the change in polyadenylation site choice for these male germ cell-specific mrnas. one surprising finding was of the existence of aberrantly polyadenylated mrnas with poly site choice occurring upstream of the reported site of polyadenylation. previous studies showed that if a pre-mrna was inefficiently polyadenylated, then rna polymerase ii read through the site of polyadenylation, producing longer transcripts. in contrast, we observed mrnas that were polyadenylated at disparate places, all upstream of the reported site and several in the luciferase coding region. we used a 3' race-based approach to look for changes in polyadenylation; this is opposed to the rnase protection assays and nuclear run-on assays previous investigators have used. because we saw this aberrant polyadenylation associated with the sv late mutant polyadenylation cassette, we believe that aberrant polyadenylation is the result of inefficient polyadenylation. it is also possible that these aberrant polyadenylation sites often occur naturally, but are only detectable in the absence of efficient polyadenylation. however, we propose that the use of a more sensitive pcr-based method has allowed us to observe previously undescribed upstream aberrant polyadenylation. finally, our data suggest the possibility that male germ cells express one or more different factors and have different sequence requirements that together alter how the polyadenylation site is chosen. other tissues may similarly have different mechanisms of polyadenylation site choice. the brain seems especially prone to contain mrnas that are alternatively polyadenylated, and our lab has identified a brain-specific form of cstf-, which may alter polyadenylation site choice in this tissue. the existence of such alternative methods of polyadenylation site choice could be used to greatly increase the protein diversity within a cell. 
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. 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. we tested whether pre-mrna sequences surrounding male germ cell-specific polyadenylation sites could be used to direct polyadenylation efficiently 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. first, male germ cells showed a lower incidence of the sequence aauaaa near the polyadenylation site choice. finally, mutation of the male germ cell-specific poly signal to a somatic poly signal resulted in more efficient polyadenylation in somatic cells. finally, a number of mrnas used a different polyadenylation site in male germ cells than in somatic cells. 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. we have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. we also developed a sensitive, 3' race-based assay to analyze polyadenylation site choice. 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 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 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.  we tested whether pre-mrna sequences surrounding male germ cell-specific polyadenylation sites 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 signal to a somatic poly 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. 