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ABSTRACTShortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.

During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2bdouble-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2ahave established roles in reproduction, the roles of many others remain to be determined.

During spermiogenesis, several proteins that are needed for the proper male germ cell differentiation are under translational control. Such proteins are expressed at very specific steps in elongating spermatids. We have recently shown that Poly(A) Binding Protein (PABP) interacting protein 2 (Paip2) is a major translational regulator involved in the maturation of male germ cellsand male fertility. Paip2A is highly expressed in the testis; it is present only in the cytoplasm of spermatids from steps 7-8 of spermiogenesis until the sperm are released. In order to identify which protein are under translational control of Paip2 regulation, we characterized the proteomic profiles of elongating spermatids from wild type (WT) mice and double knock-out (DKO) transgenic mice in which both Paip2A and Pai2B genes were abrogated. Populations containing elongating and elongated spermatids were obtained after separation on a BSA gradient with a minimum purity of 90%. Proteins were extracted and loaded on a 7% to 15% gradient acrylamide gels. After electrophoresis, the gels were cut into 15 bands and proteins were digested with trypsin. The peptides obtained were identified by mass spectrometry and analyzed using Scaffold 2.0 software.We generated a list of 632 proteins for which we could identify at least two peptides and for which we have 95% confidence. 286 proteins were consistently detected in the three WT replicates and 289 proteins in the three DKO replicates. Of these consistently detected proteins, 9 and 13 proteins were never detected in the DKO and WT, respectively. Moreover, 17 proteins present in both samples showed a significant difference with at least a 1.5 fold change in the DKO compared to the WT; 4 proteins of them were down-regulated in the DKO and 13 proteins were up-regulated. Overall, 39 proteins were differentially expressed in the DKO when compared to the WT. These proteins are involved in various cellular functions such as translation (60S ribosomal protein L13,eukaryotic translation initiation factor 4H and 4G1, elongation factor 2), metabolism (hexokinase1,hydroxyacyl-coenzyme A dehydrogenase, protein kinase C substrate 80K-H, pyridoxal-dependent decarboxylase domain-containing protein 1) and proteolysis (dipeptidylpeptidase 3, dipeptidase 3, ubiquinol cytochrome c reductase core protein 2, proteasome 26S subunit, non ATPase (PSDM1)). Interestingly, all of the proteins involved in proteolysis were down regulated in DKO; this is consistent with the abnormal maintenance of expression of PABP in late spermiogenesis. Moreover, the PSDM1 has been suggested to be present in the perinuclear-arranged manchette of rat sperm and to be responsible for the degradation of testis-specific histone in the process of chromatin packaging. This protein cannot be detected in the DKO, in which the chromatin of elongated spermatids shows abnormal vacuolization and bubble-like structures, suggesting low compaction level. Further analysis of such candidate protein will provide new insight into chromatin compaction mechanism. Supported by CIHR.(platform)

Paip2 regulates translation by promoting the dissociation of PABP (PolyA Binding Protein) from the poly(A)tails of mRNA. There are 2 homologs of Paip2,Paip2A and Paip2B, that are highly expressed in the pancreas. In the testis, Paip2A is highly expressed whereas Paip2B levels are low. During spermatogenesis, mRNAs are stored in the male germ cells as transcription is active until early spermiogenesis in round spermatids. Translation is tightly regulated during spermiogenesis since, in the absence of any transcriptional activity, several proteins are only expressed at very specific steps. Some mechanisms by which translation is regulated during the late phase of spermatogenesis have been suggested but this remains unclear. Using immunohistochemistry, we have shown that Paip2A and PABP are both specifically expressed in the testicular germ cells showing an overlapping expression during spermiogenesis. Paip2A is expressed in spermatids from steps 7-8 of spermiogenesis until the sperm are released. PABP starts to be expressed in the pachytene spermatocytes at stages 5 and its expression increases gradually in the round spermatids, to finally disappear in the elongating spermatids at step 15 of spermiogenesis. In order to understand the role of Paip2 in spermatogenesis and male fertility, we characterized a double knock-out (DKO) transgenic mouse lineage in which both Paip2A and Pai2B genes were inactivated. By continuous mating with wild type (wt) partners, we showed that DKO females presented normal fertility whereas DKO male mice were infertile. When compared to wt males, the DKO males had a 6% lower body weight as well as significantly lower epididymal (25% less) and prostatic weights (71% less). No spermatozoa could be found in the cauda epididymidis and the testis sperm head count was significantly decreased by 64%. Histological examination of the testes revealed abnormalities with vacuolization in some tubules and abnormal alignment of the spermatozoa at the lumen at stages 7-8, thus suggesting that the elongated spermatids in the DKO mouse are retained and cannot be released into the lumen properly. Moreover, we have shown an abnormal expression of PABP in the single Paip2A knock-out male germ cells where PABP is still expressed in elongated spermatids at stage 8 and staining can be found in the lumen all along the epididymis. We hypothesize that Paip2 translation factors are major players in the development of male germ cells, regulating the translation of key proteins involved in chromatin condensation and sperm release during spermiogenesis. Supported by CIHR.(platform)