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Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, is essential for spermatogenesis

The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme’s adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity.

central in the life of mRNAs by regulating stability, translation initiation and termination, and degradation [1][2][3][4]. Thus, controlling poly(A) tail length is one of the post-transcriptional regulations of gene expression. In mammalian cells, primary transcripts are generally cleaved within 30 nucleotides (nt) downstream of the polyadenylation signal, AAUAAA, followed by the addition of ~250 nt poly(A) tails by poly(A) polymerase α (PAPOLA) in a co-transcriptional manner [5,6]. Besides nuclear polyadenylation, cytoplasmic polyadenylation occurs during oocyte maturation and early embryogenesis, where transcription is entirely quiescent. Maternal mRNAs transcribed in earlier stages are deadenylated upon their exit from the nucleus (~A 10 ) and are stored in a translationally inactive state. During specific developmental stages, subsets of mRNAs undergo cytoplasmic poly(A) tail extension (~A 200 ) as a prerequisite for translational activation [7][8][9][10][11].
Spermatogenesis is a specialized cellular differentiation process that produces spermatozoa, which comprises spermatogonial proliferation, two meiotic divisions, and drastic morphological remodeling during spermiogenesis, the haploid phase of spermatogenesis. This process is accomplished by a controlled, stage-specific gene expression program regulated at the transcriptional, post-transcriptional, and translational levels [12][13][14][15][16][17]. At least two distinct patterns of poly(A) tail length changes have been described during spermatogenesis.
Haploid-specific mRNAs encoding structural proteins required for the formation of flagella and highly compacted nuclei are transcribed in round spermatids and are stored as translationally inert messenger ribonucleoprotein particles (mRNPs) with long poly(A) tracts (~A 180 ) for several days. Subsequent translational activation of the mRNAs during later spermiogenesis accompanies the generation of partially deadenylated species ranging from A 150 to A 30 [18,19]. Conversely, poly(A) tails of some mRNAs are 50-150 nt longer in round spermatids than in meiotic pachytene spermatocytes [13,[20][21][22][23].
We previously reported that ablating the testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP results in impaired spermiogenesis and male infertility, which can be recovered by transgenic re-expression [23,24]. This enzyme is responsible for cytoplasmic poly(A) tail extension of certain transcription factor mRNAs in round spermatids [23]. However, additional poly(A) tail elongation appeared not to be responsible for either stability or translational efficiency of the substrate mRNAs ( Fig. 1) [23]. These findings prompted the hypothesis that PAPOLB polyadenylation activity is dispensable for spermatogenesis. To address this, we produced and analyzed PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant.

Plasmid construction
Expression plasmids encoding N-terminally FLAG-and HA-tagged PAPOLB and polyadenylation-defective PAPOLB D114A mutant, in which Asp at position 114 (one of the three putative catalytic Asp residues) was replaced with Ala, were constructed as follows. The cDNA fragment encoding the entire open reading frame of PAPOLB was amplified by polymerase chain reaction (PCR) using the primers 5'-GGAATTCATGATGCCATTTGCGTGACC-3' and 5'-TCCTCGAGCTAGACTCCTAGTATAGGATTGG-3', and the cloned mouse cDNA as a template [22]. After digestion with EcoRI and XhoI, the fragment was inserted into pcDNA3-FLAGHAN, which is designed to express the protein with N-terminal FLAG and HA tags (PAPOLB/pcDNA3-FLAGHAN). The plasmid encoding FLAG-and HA-tagged PAPOLB D114A (PAPOLB D114A /pcDNA3-FLAGHAN) was generated by PCRbased mutagenesis using the primer pair (mutated nucleotides are underlined): 5'-CGAAAGGTGCAGCTATCGATGCTTTG-3' and 5'-CAAAGCATCGATAGCTGCACCTTTCG-3' and PAPOLB/ pcDNA3-FLAGHAN as a template.

Immunoblot analysis
Testicular tissues or purified germ cells were homogenized at 4°C in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, supplemented with 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM DTT, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), using a Teflon-glass homogenizer (750 rpm, 10 strokes). After incubation at 4°C for 4 h, the homogenates were centrifuged at 13,400 × g for 10 min at 4°C. The supernatant solutions were used as protein extracts. Protein concentration was determined by using a Coomassie protein assay reagent kit (Thermo Fisher Scientific). Protein samples (5 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). After blocking with 2% skim milk or gelatin, the blots were probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). The immunoreactive bands were visualized by an ECL or an ECL Prime Western blot detection kit (GE Healthcare).

Histological analysis
Testicular and epididymal tissues were fixed with Bouin's fixative and embedded in paraffin. Paraffin sections (4 µm thick) were prepared by a MICROM HM340E (Microedge Instruments, White Rock, BC, Canada), mounted on slides, deparaffinized in xylene, and hydrated in a graded ethanol series. After staining with hematoxylin and eosin (Wako, Osaka, Japan), the slides were observed under a DM IRBE microscope (Leica Microsystems, Wetzlar, Germany).

Statistical analysis
Data are presented as mean ± SEM (n ≥ 3), unless stated otherwise. The Student's t-test was used for statistical analysis; significance was assumed at P < 0.05.

Ethics statement
All animal experiments were approved and performed in accordance with the Guide for the Care and Use of Laboratory Animals at the University of Tsukuba (approval numbers 14-022 and 15-015).

Results
We previously found that PAPOLB catalyzes additional poly(A) tail extension of specific transcription factor mRNAs in round spermatids [23]. To examine whether cytoplasmic polyadenylation by PAPOLB is specific for transcription factor mRNAs, we analyzed the lactate dehydrogenase C subunit (Ldhc) and two forms of ornithine decarboxylase (Odc) mRNAs. Northern blot analysis showed that these mRNAs are ~100 nt longer in round spermatids than in pachytene spermatocytes of wild-type mice (Papolb +/+ , +/+), as described previously [21,28]. In contrast, no (for Ldhc) or slight (for Odc) increase in mRNA length was observed in round spermatids of PAPOLB-null mice (Papolb -/-, -/-). After RNase H digestion in the presence of oligo(dT) 15 , each mRNA decreased in length and their size was uniform among four cell types, indicating that the size differences are due to poly(A) tail elongation (Fig. 1A). Extensive analysis revealed that the ribonuclease H2 A subunit (Rnaseh2a), a male germ cell-specific paralog of general transcription factor IIA α/β subunit (Gtf2a1l/Alf/TfIIaτ), and TATA-binding protein-associated factor subunit 9 (Taf9) mRNAs are also regulated by PAPOLB. These results indicate that PAPOLB is capable of extending poly(A) tails of different mRNA classes. The levels of substrate mRNAs in round spermatids of Papolb -/mice were comparable to those in Papolb +/+ mice, despite the incomplete poly(A) tail extension (Fig. 1A). We next examined protein levels encoded by two previously identified target mRNAs (Taf10 and Tbpl1) and Rnaseh2a mRNA. Though it has long been believed that there is a positive correlation between poly(A) tail length and translational efficiency [9], the amount of TAF10, TBPL1, and RNASEH2A protein in round spermatids was similar between Papolb +/+ and Papolb -/mice (Fig. 1B). Sucrose gradient analyses also showed that additional polyadenylation of Tbpl1 and Rnaseh2a mRNAs in round spermatids did not greatly increase the ratio of mRNAs associated with translationally active polyribosomes ( Supplementary Fig. 1: online only), as was reported for Ldhc mRNA [21]. Collectively, these results suggest that additional poly(A) tail extension by PAPOLB is not responsible for enhancing either stability or translation of the mRNAs examined.
The results described above led us to speculate that PAPOLB regulates spermiogenesis independently of its polyadenylation activity. To address this possibility, we sought to examine whether PAPOLB-null phenotypes can be recovered by transgenically introducing polyadenylation-defective PAPOLB. First, we tested whether Asp at residue 114 (Asp 114 ), one of three putative catalytic Asp residues conserved in the DNA polymerase β-like nucleotidyltransferase superfamily [22], is essential for polyadenylation activity. The N-terminally FLAG-and HA-tagged PAPOLB D114A mutant (hereafter, abbreviated as PAPOLB D114A ), where Asp 114 was mutated to Ala, was assayed for non-specific polyadenylation activity [22]. When the same amount of in vitro translation product was analyzed, wild-type PAPOLB was capable of adding AMP residues on oligo(A) 12 , whereas PAPOLB D114A mutant only showed activity comparable to background levels ( Fig. 2A and B). We thus generated transgenic mice expressing PAPOLB D114A under control of the Papolb promoter (Fig. 2C). Immunoblot analysis of testicular extracts from two transgenic mouse lines, designated as Tg16 and Tg66, detected a 72-kDa protein band reactive to anti-HA antibody, which corresponds to FLAG-and HA-tagged PAPOLB D114A (Fig.  2D). Because Tg16 male mice produced mutant protein at levels equivalent to endogenous 70-kDa PAPOLB, we mainly used Tg16 for further experiments.
Both Tg16 male and female mice grew normally and were apparently normal in behavior, body weight, and health condition. When male fertility was tested by mating with wild-type females, the average litter size from Tg16 males (8.2 ± 0.4 pups/litter, n = 5) was comparable to that of wild-type matings (8.4 ± 0.4 pups/litter, n = 5, Fig. 3A). The testicular weights of Tg16 and wild-type littermates were also similar (90.3 ± 5.3 and 91.7 ± 4.0 mg in Tg16 and wild-type mice, respectively, n = 6, Fig. 3B). Histological analysis indicated that spermatogenesis and sperm migration, respectively, in the testis and epididymis of Tg16 mice were unaffected ( Supplementary Fig. 2: online only). Normal progression of spermatogenesis to elongating/ elongated spermatids in transgenic mice was also evidenced by the unaltered expression of postmeiotic mRNAs and the presence of translation-coupled deadenylated mRNA species (0.4 kb for Prm1 and Tnp1 and 0.6 kb for Prm2 and Tnp2, Fig. 3C) [18,19]. Furthermore, the abundance of four testis-specific proteins, PRM2, TNP2, PGK2, and AKAP4, was similar among wild-type and two transgenic mouse lines (Fig. 3D). These results indicate that the transgenic expression of PAPOLB D114A in wild-type mice has no dominant-negative effect on spermatogenesis.

Discussion
This study describes the essential role of PAPOLB polyadenylation activity in spermatogenesis. The enzyme is capable of extending poly(A) tails of not only transcription factor mRNAs but also mRNAs in other classes (Fig. 1). In addition, poly(A) tail extension is unlikely to enhance stability and translational efficiency of the substrate mRNAs examined because the levels of mRNAs and their translation products were not compromised by the absence of PAPOLB (Fig. 1). These observations raised the possibility that PAPOLB possesses a critical function besides poly(A) polymerase activity. Nevertheless, transgenic introduction of polyadenylation-defective PAPOLB D114A in PAPOLB-null mice failed to compensate for loss of endogenous PAPOLB (Figs. 4 and 5), implying that spermatogenesis indeed requires cytoplasmic polyadenylation catalyzed by PAPOLB, or at least its terminal nucleotidyltransferase activity.
Efficient translation of eukaryotic mRNAs is accomplished by cap and poly(A) tail synergy mediated by three proteins: cytoplasmic poly(A)-binding protein (PABPC) bound to poly(A) tails associates with the scaffold protein eukaryotic initiation factor (EIF) 4G, which is also capable of interacting with EIF4E, a cap-binding protein. This "closed-loop" structure is thought to be critical for efficient translation by promoting ribosome recycling and/or enhancing the interaction between initiation factors and mRNAs, as well as for protecting mRNAs from deadenylation [29][30][31][32]. Given that transcription is entirely silent during oocyte maturation and early embryogenesis, mRNAs encoding proteins required for progression of these processes are transcribed in earlier stages and are stored as deadenylated forms after transport to the cytoplasm [7][8][9][10][11]. These maternal mRNAs with short poly(A) tails (~A 10 ) are translationally incompetent, where PABPC is unable to bind mRNAs, and are later activated by cytoplasmic polyadenylation. However, poly(A) tail extension by PAPOLB differs from the case during oocyte maturation and early embryogenesis in that the substrate mRNAs identified to date already possess poly(A) tract lengths (~100 nt) sufficient for PABPC-binding. Additional poly(A) tail extension by PAPOLB had no impact on translation and steady-state level of the mRNAs and is thus considered to be non-specific or a side effect in view of function (Fig. 1) [23]. This is consistent with the recent findings that the median length of poly(A) tails is 50-100 nt in mammalian cells, which is shorter than previously thought (150-250 nt), and that additional poly(A) tail elongation does not further contribute to translational enhancement except on maternal mRNAs [33][34][35]. Thus, if cytoplasmic polyadenylation by PAPOLB governs spermiogenesis, it is expected that the "true" target(s) possesses maternal mRNAlike short poly(A) tail(s). It is also conceivable that PAPOLB, like noncanonical poly(A) polymerase PAPD4/GLD-2, is involved in 3'-end monoadenylation of certain microRNAs, thereby regulating their stability [36,37].
It is interesting to note that the poly(A) tails of some mRNAs, including odc (Fig. 1A) and Taf10 [23], are slightly longer in round spermatids than in pachytene spermatocytes of PAPOLB-deficient mice, but are shorter in round spermatids of wild-type mice. These observations imply that poly(A) tail extension of the mRNAs is carried out not only by PAPOLB but also by another poly(A)  polymerase, such as PAPD4 [38]. Indeed, poly(A) tails of Acr, Acrbp-W, Acrbp-V5, and Zpbp1 mRNAs were still elongated in round spermatids even in the absence of PAPOLB (Supplementary Fig. 4: online only) [23]. Identifying the polyadenylation pathway responsible for these processes will be helpful to understand poly(A) tail dynamics during mammalian spermatogenesis. Although PAPOLB polyadenylation activity is most likely essential for spermatogenesis, we cannot rule out the possibility that Asp 114 of PAPOLB is involved in another yet unknown activity, as is the case for protein arginine methyltransferase 8 (PRMT8), in which Ser 120 is required for both arginine methylation and phosphatidylcholine hydrolysis activities [39]. At any rate, further studies, including the identification of the "true" target(s), are necessary to elucidate PAPOLB function.  15 , and subjected to Northern blot analysis with the cDNA probes indicated. (B) Expression of testis-specific mRNAs. Testicular total RNAs were analyzed by Northern blotting using the cDNA probes indicated. Actb was used as a control. (C) Translation of testis-specific mRNAs. Testicular protein extracts were subjected to immunoblot analysis with the antibodies indicated. ACTB served as a control.