Precursor RNA processing 3 is required for male fertility, and germline stem cell self-renewal and differentiation via regulating spliceosome function in Drosophila testes

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. Our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes.

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNps) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the prp complex, is essential for male fertility, and germline stem cell selfrenewal and differentiation, via regulating the spliceosome function in Drosophila testes.
Spermatogenesis is highly conserved and widespread in eukaryotes, from Drosophila to humans [1][2][3] . Mutants of boule and many other genes exhibit similar testicular phenotypes in humans and Drosophila 4,5 . Drosophila works as an excellent animal model to study male fertility [6][7][8] . Adult Drosophila testes contain different stages of germ cells from spermatogonia to mature sperm 8,9 . At the head area of the testis, germline stem cells (GSCs) divide to a new stem cell and a gonialblast, which could proliferate and differentiate into spermatocytes 10 . The gonialblast goes through mitosis process to form a 16-cell spermatogonia cluster, connecting by ring canals and a branched (2019) 9:9988 | https://doi.org/10.1038/s41598-019-46419-x www.nature.com/scientificreports www.nature.com/scientificreports/ fusome 11,12 . Cyst stem cells (CySCs) can also differentiate to mature cyst cells, which help to maintain the growth of germ cells 3 .
Early germ cells in fly testes are tightly controlled by niche signals 1 . Hub cells secrete the unpaired (Upd) protein, which activates the Janus kinase signal transducer and activator transcription (JAK-STAT) pathway in both GSCs and CySCs 13,14 . Hedgehog (Hh), also secreted in hub cells, is required for CySCs to maintain their pluripotency by Hh signaling pathway 15,16 . Two somatic expressed bone morphogenetic (BMP)-like molecules, Decapentaplegic (Dpp) and Glass bottom boat (Gbb), are essential for the GSC maintenance [17][18][19] . Bag-of-marbles (Bam) is a significant differentiation factor, which could be repressed by BMP signaling 18,19 . Benign gonial cell neoplasm (Bgcn) is identified as the regulatory factor of Bam, and controls the spermatogonia transition from self-renewal to differentiation [18][19][20] . Loss of function of bam or bgcn gene lead to differentiation defects with extensive accumulation of undifferentiated germ cells 20,21 .
Most genes in eukaryotic genomes require the spliceosome to remove the introns from nuclear pre-mRNAs. The major spliceosome (U2 type) and the minor spliceosome (U12 type) are two main types of spliceosomes 22,23 . RNA splicing is conserved and is catalyzed by the spliceosome, containing small nuclear ribonucleoprotein particles (snRNPs) and many non-snRNP protein factors 24 . Comparison of the spliceosome protein composition by mass spectrometry (MS) identified more than 120 proteins in humans and Drosophila, indicating the evolutionarily conserved composition of the mRNA spliceosome 25 . Recently evidence demonstrated that U2A, a key component of the spliceosome, is required for male fertility and regulates the transition of germ cells from proliferation to differentiation. A point mutant of human SNRPA1 in flies also led spermatogonial differentiation defects 26 .
In yeast, precursor RNA processing 3 (Prp3) and several other U4/U6 snRNP-associated splicing genes have been identified by genetic screening for RNA synthesis 27,28 . However, few reports indicate the biological function of Prp3 in animal models. In our previous screen, Prp3 was identified as a male GSC regulatory factor that played roles in Drosophila testes 29 . Here, we further investigated roles of Prp3 in male fertility, and the self-renewal and differentiation of germline stem cells in Drosophila.

Results
Prp3 is crucial for male fertility. To explore the role of Prp3 in Drosophila, we evaluated the male fertility rate in control and Prp3 RNAi males. We used three Gal4s to knock down Prp3 gene: Nos-Gal4 mainly works in early germ cells especially in GSCs, Bam-Gal4 is a spermatogonia driver, and Tj-Gal4 is considered as a cyst cell driver in Drosophila testes 29 . When we knocked down Prp3 driven by Nos-Gal4, males were totally sterile (n = 93) compared with the wild-type control (96.63% fertile, n = 89) (Fig. 1A). We further analyzed the function of the Prp3 gene by using Bam-Gal4 and Tj-Gal4. Interestingly, males with Bam > Prp3 RNAi (2.86% fertile, n = 70) and Tj > Prp3 RNAi (8.75% fertile, n = 80) both lost their fertility ability (Fig. 1A). Taken together, our data suggested that Prp3 is required for male fertility in Drosophila.
Prp3 is required for GSC maintenance. We next dissected control and Nos > Prp3 RNAi testes, and stained them with several markers to label the different kind of cells in testes. Somatic cells, including hub cells and cyst cells, can be tagged by DE-cadherin (DE-cad). Fasciclin III (Fas III) labels hub cells, 1B1 labels fusomes in the germ cell cyst and Vasa labels germ cells in the testis. Vasa-positive germ cells closely connected to the hub cells are GSCs and Vasa-negative cells linked together with the hub cells are CySCs 3,30 . In the control testes, point fusomes existed in the early germ cells at the tip of testes and developed into branch fusomes in the differentiated germ cells (Fig. 1B). However, 1B1-positive fusomes and Vasa-labeled germ cells were totally absent in the Nos > Prp3 RNAi testis (Fig. 1C), which meant that GSCs and differentiated germ cells were not maintained. Our results indicated that Prp3 is required for GSC survival.
Prp3 is required for CySC maintenance and regulates GSC differentiation. Next, we wondered whether Prp3 plays similar role in CySCs and their differentiated cyst cells. In Tj > Prp3 RNAi testes, Zn finger homeodomain 1 (Zfh-1)-positive CySCs and eyes absent (Eya)-positive cyst cells were absent when comparing with control testes ( Supplementary Fig. 1A,B). Thus, Prp3 is also essential for cell survival of CySCs and mature cyst cells.
Surprisingly, undifferentiated cells accumulated and tumors formed in Tj > Prp3 RNAi testes. We hypothesized that the cells accumulated in Tj > Prp3 RNAi testes may be the undifferentiated germ cells without niche control. To test this hypothesis, we stained the testis and found that the undifferentiated cells could be labeled by anti-Vasa antibodies and accumulated without normal niche cells (Fig. 1D,E and Supplementary Fig. 1C,D). Moreover, only point fusomes existed (white arrowheads) and no branched fusomes were observed in Tj > Prp3 RNAi testes (Fig. 1E"); however, branched fusomes (yellow arrowheads) were observed in differentiated germ cell cysts in the control testes ( Fig. 1D"). We further stained with phosphor histone H3 (PH3), a proliferation marker, to test the cell fate of these undifferentiated germ cell cysts. These results showed that the undifferentiated germ cells in Prp3 RNAi testes could proliferate and maintain themselves without hub cells (Fig. 1D,E and Supplementary Fig. 1C,D). Taken together, our data indicated that knockdown of Prp3 gene by Tj-Gal4 could cause GSCs differentiation defects and tumor formation.
Prp3 deficiency in spermatogonia causes spermatogonia differentiation defects. Bam is a key differentiation factor in Drosophila testes. Knocking down of bam in spermatogonia severely affected germ cell differentiation. Notably, undifferentiated germ cells accumulated and obtained the ability to proliferate by themselves in Bam > bam RNAi testes ( Fig. 2A,B,F,G).
We also questioned the role of Prp3 in spermatogonia differentiation. We next knocked down Prp3 in spermatogonia driven by Bam-Gal4. In Bam > Prp3 RNAi testes, point fusomes were significantly increased ( Fig. 2A,C,E) with statistical differences (all differences relative to control), and PH3-positive cells could also www.nature.com/scientificreports www.nature.com/scientificreports/ be observed distant to the hub cells (Fig. 2F,H). Interestingly, Prp3 mimicked the phenotype of bam in the spermatogonia transition from self-renewal to differentiation.
Previous study indicated that heterozygous mutation of bam did not lead to dramatic differentiation defects in Drosophila testes 7 . However, phenotype of differentiation defects were obviously enhanced in Bam > Prp3 RNAi; Δ86/+ testes (more point fusomes and accumulated undifferentiated germ cells), compared with Bam > Prp3 RNAi ( Fig. 2C-E,H,I).
In control testes, many clusters of elongated spermatids (39.89 ± 1.80) could be observed, while only few clusters of elongated spermatids were observed in Bam > Prp3 RNAi (2.26 ± 0.45), Bam > bam RNAi (1.12 ± 0.14), and Bam > Prp3 RNAi; Δ86/+ (1.16 ± 0.18) testes ( Fig. 2J-N, all differences relative to control). Phase-contrast view allowed us to identify spermatogenic cells at different stages (spermatogonia, spermatocytes, round spermatids, elongated spermatids, and mature sperm) in wild-type testes (Fig. 3A). Nonetheless, only spermatogonia existed and accumulated at the tip of testes in Bam > Prp3 RNAi and Bam > Prp3 RNAi; Δ86/+ cells compared with those of the control (Fig. 3B-D). These results suggested that the differentiation disorder of early germ cells caused by loss of Prp3 influenced spermatogenesis, ultimately leading to male infertility.

Prp19 and Prp8 are essential for GSC self-renewal and differentiation in the Drosophila testis.
The Prp complex is a large protein family that participates in RNA splicing. We next tested several other www.nature.com/scientificreports www.nature.com/scientificreports/ components of the Prp complex, and found that Prp19 and Prp8 have similar functions in Drosophila testes. When we knocked down Prp19 and Prp8 using Nos-Gal4, males were totally sterile (n = 63 for Prp19; n = 58 for Prp8). Moreover, males were totally sterile in Tj > Prp19 RNAi (n = 44) flies and partially infertile in Tj > Prp8 RNAi (10.53% fertile, n = 57) flies (Supplementary Table S1). DE-cad labels hub cells and cyst cells, and Vasa labels germ cells in the testis. For the abnormal structure of testes, germline stem cells adjacent to the hub cells and subsequently differentiated germ cells lost. After knockdown of Prp19 and Prp8 with Nos-Gal4, normal structure of testes disappeared (Supplementary Table S2), and Vasa-positive germ cells were absent (Fig. 4A,B) compared with those of the control (Fig. 1B). Moreover, only point fusomes existed among undifferentiated germ cells in Tj > Prp19 RNAi and Tj > Prp8 RNAi testes (Fig. 4C,D) when comparing with the control testes (Fig. 1D). These data suggested that Prp19 and Prp8 are also essential for GSC self-renewal and differentiation in fly testes.

Prp3 regulates proliferation and cell survival in Drosophila S2 cells.
To explore the function of Prp3 in apoptosis and proliferation, we further downregulated Prp3 expression using two siRNAs (Prp3 siRNA-354 and Prp3 siRNA-1660) by in vitro approaches. Our results indicated that siRNA-mediated knockdown of Prp3 in Drosophila S2 cells reduced the expression of the Prp3 mRNA (Fig. 5A).
In S2 cells, PH3-positive cells were significantly decreased in Prp3 siRNA (150 nmol) S2 cells ( Supplementary  Fig. S2). Moreover, when Prp3 was knocked down in these cells using Prp3 siRNA (150 nmol), TUNEL-positive cells were dramatically increased (Fig. 5B,C), indicating that Prp3 was essential for cell survival. Similar results were obtained by flow cytometry, which showed that the ratios of apoptotic and necrotic cells significantly increased in Prp3 siRNA S2 cells (Fig. 5D,E). Next, we used a CCK-8 kit to detect whether the growth of Prp3 siRNA-treated S2 cells was affected, and found that knockdown of Prp3 in S2 cells dramatically reduced cell proliferation, compared with control (Fig. 5F). Taken together, these results indicated that the Prp3 knockdown results in decreased proliferation and increased cell death in Drosophila.
Prp3 regulates the expression level of spliceosome subunits in Drosophila S2 cells. To further investigate whether Prp3 affects spliceosome, we measured the expression level of major spliceosome subunits of www.nature.com/scientificreports www.nature.com/scientificreports/ the Prp complex and Sm complex. Surprisingly, the qRT-PCR results showed that spliceosome subunits, including key components of the Prp complex (Prp18, Prp19, and Prp8) and Sm complex (SmB, SmD1, SmE, SmF, and SmG), were all downregulated in Prp3 siRNA S2 cells (Fig. 6). These results indicated that Prp3 may be a key protein that could regulate the expression level of spliceosome subunits.

Discussion
The spliceosome is a fundamental element for the constitutive and alternative splicing of pre-mRNA to generate mature mRNA. The U4/U6-associated splicing factor, Prp3, is conserved in humans and Drosophila with 45% sequence identity. Prp3 is predominantly expressed in Drosophila ovaries and localizes in the nuclei of the female reproductive cells 31 . The other spliceosomal gene prp22 (pea) is required for chromatin dispersal in nurse cell nuclei during oogenesis 32 . However, the biological function of Prp3 in male fertility and stem cell niche remain to be determined.
Here, we systematically analyzed the mechanism and function of Prp3 in Drosophila using in vivo and in vitro approaches. Our results indicated that Prp3 plays key roles for the germline stem cell niche in the Drosophila testis, and controls the GSC self-renewal and differentiation processes. Our data provided a model of Prp3 which  www.nature.com/scientificreports www.nature.com/scientificreports/ functions in the germline stem cell niche, that maintenance defects of GSCs caused abnormal structure of testes and loss of germ cells, while maintenance defects of CySCs led to dysfunction of somatic stem cells and, followed by early germ cell differentiation defects with non-cell autonomous function.
The S2 cell line was derived from a primary culture of late stage (20-24 hours old) Drosophila melanogaster embryos. Although the S2 cell line is not a germ cell line, but it is a classical cell model in Drosophila. Knockdown of Prp3 in vitro decreased the proliferation ability and dramatically increased the cell death ratio in S2 cells, which imitated the phenotype in the germline stem cell niche. More importantly, in vitro assays provided evidence that knockdown of Prp3 might destroy the major structure of the spliceosome and affect its function by regulating major subunits of the Prp and Sm complexes. Although there are subtle differences in some biological events, the evidence indicated that the spliceosome complex may play critical roles in azoospermia and germ cell tumor formation. Further assessment using a germline stem cell model will be investigated in future studies.
By querying The Drosophila Interactions Database (DroID), we found that Prp3 could bind with many snRNPs and Prp factors, such as SmD2, LSm3, U2af50, and U4-U6-60K 33 . Moreover, the Prp19 complex regulates the ubiquitination modification of Prp3 34 . On the other hand, Prp8 can also recognize the ubiquitination chains of Prp3 and stabilizes the U4/U6.U5 tri-snRNP 35 .
In our study, we observed that Prp3, Prp8, and Prp19 played diverse roles in GSCs and CySCs. GSCs lacking Prp3, Prp8, and Prp19 could not maintain themselves while CySCs lacking Prp3, Prp8, and Prp19 are not RNAi. Student's t test was used for the statistical analysis. *represents for P value < 0.05, **represents for P value < 0.01, ***represents for P value < 0.001. Error bars represent SEM. Scale bar: 30 μM. (2019) 9:9988 | https://doi.org/10.1038/s41598-019-46419-x www.nature.com/scientificreports www.nature.com/scientificreports/ sufficient for GSCs to differentiate to terminal germ cells. We hypothesized that this is caused by diverse splicing of pre-mRNA targets. However, the effector functions of Prp3, Prp8, and Prp19 in GSCs and CySCs remain poorly understood. Screening for the downstream factors and splicing targets of Prp3, Prp8, and Prp19 in the germline stem cell niche will provide new insights for understanding the mechanism of azoospermia and germ cell tumor formation. Fly crosses. We used UAS/GAL4 system to knock down shRNA-targeted genes in specific cell populations.

Materials and Methods
All UAS-RNAi transgenic lines were obtained from the THFC 29 . GAL4 males are crossed with UAS-RNAi virgin females raised at 25 °C. In the next generation, we selected males with both GAL4 and UAS-RNAi elements. Immunofluorescence. Fly testes were dissected and fixed for 30 min in 4% paraformaldehyde. After washing three times in 1x PBS with 0.1% Triton X-100 (PBST) and blocking for 1 hr in 5% bovine serum albumin, samples were incubated with primary antibodies overnight at 4 °C. After washing three times for 10 min in 0.1% PBST, the samples were incubated for 1 hr with secondary antibodies at room temperature followed by three times washing in 0.1% PBST. Testes were then stained with Hoechst 33342 (1.0 mg/ml, Invitrogen) for 5 min before mounting. S2 cells were cultured for 24 hours, and immunostaining was carried out in the culture dish according to the protocols described above 7 .
The antibodies used were as follows:  *represents for P value < 0.05, **represents for P value < 0.01, ***represents for P value < 0.001. Error bars represent SEM.