(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



RNA-binding protein Maca is crucial for gigantic male fertility factor gene expression, spermatogenesis, and male fertility, in Drosophila

['Li Zhu', 'Department Of Biological Chemistry', 'Johns Hopkins University School Of Medicine', 'Baltimore', 'Maryland', 'United States Of America', 'Ryuya Fukunaga']

Date: 2021-08

During spermatogenesis, the process in which sperm for fertilization are produced from germline cells, gene expression is spatiotemporally highly regulated. In Drosophila, successful expression of extremely large male fertility factor genes on Y-chromosome spanning some megabases due to their gigantic intron sizes is crucial for spermatogenesis. Expression of such extremely large genes must be challenging, but the molecular mechanism that allows it remains unknown. Here we report that a novel RNA-binding protein Maca, which contains two RNA-recognition motifs, is crucial for this process. maca null mutant male flies exhibited a failure in the spermatid individualization process during spermatogenesis, lacked mature sperm, and were completely sterile, while maca mutant female flies were fully fertile. Proteomics and transcriptome analyses revealed that both protein and mRNA abundance of the gigantic male fertility factor genes kl-2, kl-3, and kl-5 (kl genes) are significantly decreased, where the decreases of kl-2 are particularly dramatic, in maca mutant testes. Splicing of the kl-3 transcripts was also dysregulated in maca mutant testes. All these physiological and molecular phenotypes were rescued by a maca transgene in the maca mutant background. Furthermore, we found that in the control genetic background, Maca is exclusively expressed in spermatocytes in testes and enriched at Y-loop A/C in the nucleus, where the kl-5 primary transcripts are localized. Our data suggest that Maca increases transcription processivity, promotes successful splicing of gigantic introns, and/or protects transcripts from premature degradation, of the kl genes. Our study identified a novel RNA-binding protein Maca that is crucial for successful expression of the gigantic male fertility factor genes, spermatogenesis, and male fertility.

Sperm is produced from germline cells via the process called spermatogenesis, during which a special gene expression program dedicated to this process operates. In fruit fly Drosophila, extremely large male fertility factor genes on Y-chromosome containing mega-base-sized introns are expressed only during spermatogenesis, and their successful expression is crucial for spermatogenesis. However, expressing such large genes must be very challenging for cells and the molecular mechanisms that ensure their successful expression remain unknown. In this study, we identified a novel RNA-binding protein encoded in the gene CG5213 that is required for this process. We created mutant flies lacking this protein using a genome-editing technique, and investigated them using genetic, microscopic, molecular, transcriptomic, and proteomic approaches. We found that this RNA-binding protein is crucial for successful expression of the extremely large male fertility factor genes, spermatogenesis, and male fertility. We named the CG5213 gene maca, after the plant maca grown in the high Andes mountains whose root has been traditionally believed and used to increase sperm quality and promote male fertility. By identifying the novel RNA-binding protein Maca, our studies give significant insight into how the special gene expression program operates during spermatogenesis.

Funding: This work was supported by the grants from the National Institutes of Health [R01GM116841] and Johns Hopkins University Catalyst Award to RF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Zhu, Fukunaga. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we hypothesized that there may be a previously uncharacterized RNA-binding protein that is important for the testis-specific gene expression program. We predicted that such an RNA-binding protein, if any, is expressed exclusively in testes. We found that the gene CG5213, located on the right of the third chromosome, meets these criteria: it encodes a novel RNA-binding protein with two RNA recognition motifs (RRMs) ( Fig 1C ) and its mRNA is expressed almost exclusively in adult testes ( Fig 1D ). We genetically investigated the functions of CG5213 and revealed that it is required for spermatogenesis and male fertility in Drosophila. We discovered that the CG5213 protein is expressed in spermatocytes, resides in the nucleus enriched at Y-loop A/C, and is required for successful expression and splicing of the kl-2, kl-3, and kl-5 transcripts. We named the CG5213 gene maca, after the plant maca grown in the high Andes mountains whose root has been traditionally believed and used to increase sperm quality and promote male fertility.

In spermatocytes, kl-3, kl-5, and ks-1 form lampbrush-like nucleoplasmic structures named Y-loops [denoted as loops A (kl-5), B (kl-3), and C (ks-1) ( Fig 1B ), analogous to the lampbrush loops of amphibian oocytes [ 33 ]. Y-loop structures reflect the robust transcription of underlying genes in spermatocytes. Each loop consists of a DNA axis associated with huge repetitive RNA transcripts, which are in turn associated with large amounts of proteins [ 33 – 35 ].

The ~40 Mb Y chromosome of Drosophila is required only for male fertility, but not fly viability, mostly (~80%) comprised of repetitive sequences (primarily short tandem repeat satellite DNAs), and entirely heterochromatic [ 17 – 19 ]. Because of the extremely repetitive nature, the genome DNA sequence of most of the Y chromosome is not fully determined yet and available genome sequences of the Y chromosome contain many gaps. Despite the considerable size of the Y chromosome, it has only 16 known protein-coding genes. The Y chromosome contains six loci called male fertility factors that are essential for spermatogenesis and male fertility ( Fig 1B ) [ 18 , 20 – 22 ]. Four (kl-5, kl-3, kl-2, and kl-1) are on the long arm of the Y and two (ks-1 and ks-2) are on the short arm. kl-2, kl-3, and kl-5 encode dynein proteins that are crucial components of the axoneme, the microtubule-based cytoskeletal structure that forms the core of a sperm flagellum [ 23 – 28 ]. The kl-2, kl-3, and kl-5 genes are transcribed during the spermatocyte growth and their mRNAs form cytoplasmic ribonucleoprotein (RNP) granules called kl-granules in late-stage spermatocytes [ 29 ]. kl-2, kl-3, and kl-5 mRNAs are suggested to start being efficiently translated only when axoneme elongates in the sperm elongation process in spermiogenesis. The male fertility factor genes including kl-2, kl-3, and kl-5 contain gigantic, megabase-sized introns rich in repetitive, satellite DNA. For example, kl-3 gene spans at least 4.3 Mb while its coding sequence is only ~14 kb [ 18 , 30 , 31 ]. Their gene sizes and structures are reminiscent of those of human Dystrophin, a causative gene for Duchenne Muscular Dystrophy, which spans ~2.2 Mb with gigantic introns rich in repetitive DNA and only ~11 kb coding sequence [ 32 ]. Transcription of these extremely large genes and processing their transcripts including splicing must be challenging for cells. There must be unknown special molecular mechanisms and factors that enable their efficient, precise, and regulated transcription and processing.

After their G2 growth phase, spermatocytes divide twice by meiosis and then differentiate into 64 interconnected haploid spermatids. After 64 interconnected spermatids elongate, they are separated into individual spermatids by individualization complexes (ICs) in the sperm individualization process ( Fig 1A ). Individualization complexes form around the sperm nuclei and move processively in a highly coordinated manner from the heads to the tips of the sperm tails along the spermatid bundle, removing excess cytoplasm and unneeded organelles and encasing each sperm cell in its own plasma membrane. The individualized, mature motile sperm are stored in the seminal vesicles for fertilization.

(A) Diagram of spermatogenesis in Drosophila testis. At the apical tip of testis, germline stem cells (GSCs) are attached to the hub cells (Hub). GSCs produce daughter cell gonialblasts, which become mitotically-amplifying spermatogonia that then become spermatocytes. Spermatocytes grow in size for ~90 hours and then undergo the meiotic divisions to become 64 interconnected spermatids, which are then separated into individual spermatids by individualization complexes (ICs). Individualized mature motile sperm are stored in seminal vesicles. (B) Diagram of the Drosophila Y chromosome. Locations of the 6 male fertility factor genes (cyan. The transcription directions of the encoded genes are indicated by blue arrows), regions enriched for satellite DNA (green bars), and the Y-loop forming regions (black bars) with associated satellite DNA sequences are shown. (C) Domain structures of Drosophila Maca (Maca/CG5213), and its mutants, Maca null and Maca R1 . (D) maca mRNA expression pattern. Data are obtained from http://flybase.org/reports/FBgn0038345 . (E) Western blots of dissected fly tissues. (F) Western blots of testis lysates.

Genetically well-tractable Drosophila has been an excellent model system for spermatogenesis studies [ 13 – 16 ]. The Drosophila testis, where spermatogenesis occurs, is a coiled tube with a closed apical end and a basal end that connects to the seminal vesicle. The germline stem cells (GSCs) reside at the apical tip attached to the hub cells and differentiating germ cells are gradually displaced distally ( Fig 1A ). GSCs divide asymmetrically to produce a self-renewed GSC and a daughter cell that undergoes differentiation and becomes 16 spermatogonia by four rounds of synchronous mitotic divisions. The 16-cell spermatogonia then enter the meiotic S phase and become spermatocytes. Spermatocytes grow in size during their G2 growth phase, which spans as long as ~90 hours. During the spermatocyte growth, the homologous chromosomes pair and partition into individual chromosome territories, and almost all genes encoding proteins for later meiotic and post-meiotic processes (spermiogenesis) are transcribed. These transcripts are stored without being efficiently translated until their protein activities are required later for meiosis and spermiogenesis. Thus, transcriptional and post-transcriptional events during the spermatocyte growth phase are crucial and must be spatiotemporally highly regulated.

Spermatogenesis is the highly conserved and tightly regulated process where diploid germline stem cells develop into mature, haploid sperm capable of fertilizing oocytes. Spatiotemporally highly regulated gene expression is crucial during spermatogenesis, and transcriptional and post-transcriptional regulation by RNA-binding proteins play key roles. Human male sterility results from abnormal spermatogenesis and is mostly due to chromosomal alterations, Y chromosome microdeletions, and related gene mutations. Mutations in the germline-specific DAZ (Deleted in AZoospermia) family of RNA-binding proteins, which contain a highly conserved RNA recognition motif (RRM) and are crucial regulators of gene expression during spermatogenesis, impair spermatogenesis and cause human male sterility [ 1 – 12 ].

Results

Maca is exclusively expressed in testes We generated a polyclonal anti-Maca antibody against a recombinant full-length Maca protein and examined Maca protein expression in Drosophila tissues using hand-dissected control (w1118) fly samples. We found that Maca protein is expressed exclusively in adult testes (Fig 1E).

maca mutant flies To study biological and molecular functions of Maca in vivo, we created two maca mutant alleles, macanull and macaR1 by introducing deletions within the Maca coding region using a CRISPR/Cas9 genome editing system (Fig 1C) [36]. The macanull allele has a 5-nt long deletion before the first RRM (deletion of 135–139 nucleotide residues in the Maca coding sequence (CDS)), which caused a translation frameshift and produced a premature stop codon resulting in encoding an N-terminal 45 amino acid (aa) fragment of Maca followed by additional 8 aa (total 53 aa, 5.7 kDa. Fig 1C). This short fragment is unlikely to have any functions and we consider this allele as null. The macaR1 allele has a 4-nt long deletion after the first RRM (deletion of 353–356 nucleotide residues in the Maca CDS), causing a translation frameshift and producing a premature stop codon. The macaR1 allele encodes an N-terminal 117 aa fragment of Maca followed by additional 37 aa (total 154 aa, 17.5 kDa. Fig 1C). Both macanull and macaR1 homozygous mutant flies were viable, showing that Maca is dispensable for fly viability. To validate the maca mutant fly strains and the Maca antibody that we created, we performed Western blots of testis lysates from the maca mutant flies using the anti-Maca antibody. In testis lysates of wild-type (maca+/+) and heterozygous controls of macanull and macaR1 (macanull/+ and macaR1/+), the full-length Maca protein was detected as expected (Fig 1F). The full-length Maca protein was not detected in the testis lysates from the homozygous mutants of macanull and macaR1 (macanull/null and macaR1/R1). These results validated our maca mutant fly strains and anti-Maca antibody. No smaller proteins corresponding to the Maca fragments were detected in the macanull and macaR1 strains (macanull/+, macanull/null, macaR1/+, and macaR1/R1). Therefore, the Maca fragments encoded in these two mutant alleles are likely unstable or not expressed.

maca is essential for male fertility Maca’s testis-specific expression (Fig 1D and 1E) suggested that Maca plays an important role in testes. To test this, we performed fertility assays to determine whether maca is required for male fertility. Hundreds of progenies were obtained when the control flies (maca+l+, macanull/+, and macaR1/+) were crossed with wild-type virgin females (Fig 2A). In contrast, no progenies were obtained at all when macanull/null or macaR1/R1 male flies were crossed with wild-type virgin females. Trans-heterozygous mutant flies macanull/Df and macaR1/Df, which have the macanull or macaR1 allele and the Df(3R)Exel6174 chromosomal deficiency allele uncovering the maca gene, also showed complete male sterility (S1A Fig). These results indicated that maca is strictly required for male fertility. To confirm that male sterility is due to loss of maca, not due to any unintended secondary mutations, we tested if a maca transgene can rescue male fertility in the macanull/null and macaR1/R1 backgrounds. We created maca transgenic rescue fly strains using a maca transgene that expresses C-terminally EGFP-fused Maca protein under control of a maca promoter. This maca-EGFP transgenic allele fully rescued male fertility in the macanull/null and macaR1/R1 backgrounds (Fig 2A). macanull/null, macanull/Df, macaR1/R1, and macaR1/Df did not exhibit any significant changes in female fertility compared with the controls (S1B and S1C Fig). We concluded that maca is strictly required for male fertility, but not for female fertility. This is consistent with the observation that Maca is expressed almost exclusively in testes but not in female flies (Fig 1D and 1E). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. maca mutant flies are male-sterile, lack mature motile sperm, and have disorganized individualization complexes. (A) Male fertility assay. The numbers of the progeny flies obtained from the crosses between test males and OregonR wild-type virgin females are shown. Mean +/- SD (n = 5 biological replicates). P-value <0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *. (B) Stereomicroscope images of dissected whole testes. Scale bars are 100 μm. (C) Confocal images of dissected seminal vesicles stained with DAPI. Mature motile sperm is absent in macanull/null and macaR1/R1. Scale bars are 50 μm. (D) Confocal images of dissected whole testis from the maca-EGFP transgenic fly. Maca-EGFP (green) and DAPI (blue). The apical tip of the testis, where the hub cells reside, is marked with an asterisk (*). Scale bar is 100 μm. (E) Confocal images of the apical region of the testis from the maca-EGFP transgenic fly. Maca-EGFP (green), Vasa (red), and DAPI (blue). The apical tip of the testis, where the hub cells reside, is marked with an asterisk (*) in the merged panel. Scale bar is 100 μm. (F) Confocal images of the apical region of the testis from the maca-EGFP transgenic fly. Maca-EGFP (green), Hts (red), and DAPI (blue). The apical tip of the testis, where the hub cells reside, is marked with an asterisk (*) in the merged panel. Scale bar is 100 μm. (G) Confocal images of the early-stage individualization complexes. Phalloidin (Actin, green), DAPI (blue). Scale bars are 10 μm. (H) Confocal images of the late-stage individualization complexes. Phalloidin (Actin, green). Scale bars are 10 μm. https://doi.org/10.1371/journal.pgen.1009655.g002

maca is essential for spermatogenesis To explore the mechanism underlying the complete male sterility in the maca mutant flies (Figs 2A and S1), we first examined whole testis morphology. We did not observe any major defects in whole testis morphology in the maca mutant flies compared with the controls (Fig 2B). Next, using DAPI staining of needle-shaped sperm nucleus and confocal imaging, we examined mature motile sperm, which are produced in testes and stored in seminal vesicles. As expected, we observed plenty of mature motile sperm in the seminal vesicles of the control flies (maca+l+, macanull/+, and macaR1/+) (Fig 2C). In contrast, the seminal vesicles of macanull/null and macaR1/R1 flies were empty lacking mature motile sperm. Plenty of mature motile sperm were observed in the seminal vesicles of macanull/null and macaR1/R1 flies with the maca-EGFP rescue transgene. Using the DJ-GFP allele, which labels mature motile sperm with bright GFP signal in the control seminal vesicles, we further confirmed the loss of mature motile sperm in the seminal vesicles of macanull/null and macaR1/R1 (S2 Fig). We concluded that maca is essential for spermatogenesis.

Maca is expressed in spermatocytes and resides in the nucleus We investigated an expression pattern of Maca protein in testes using the maca-EGFP transgenic flies. We found that Maca-EGFP is expressed in spermatocytes residing in the nucleus and becomes undetectable after spermatocytes complete their growth (Fig 2D). Next, we performed immunostaining of maca-EGFP transgenic fly testes with Vasa and Hu-li tai shao (Hts) antibodies. Maca-EGFP was expressed in spermatocytes and was localized in the nucleus while Vasa was expressed in germline stem cells and developing germline cells including spermatogonia and spermatocytes and resided in the cytoplasm and at the nuclear periphery (Fig 2E). Hts labels filamentous fusome structures connecting the dividing spermatogonia and spermatocytes (Fig 2F). Maca-EGFP started to be expressed soon after the Hts filamentous structures were formed. These results together showed that Maca-EGFP is expressed in spermatocytes and resides in the nucleus. We also transiently expressed EGFP-Maca in cultured S2 cells and found that EGFP-Maca protein is localized in the S2 cell nucleus (S3 Fig), confirming that Maca is a nuclear protein.

maca is essential for sperm individualization To examine which step in spermatogenesis is impaired in macanull/null testes, we examined the sperm individualization process [37]. Individualization complexes were properly organized in the early stage of the sperm individualization process in macanull/null testes as in the control testes (Fig 2G). However, unlike in the control testes, individualization complexes were disorganized and scattered in the late stage of the individualization process in macanull/null testes, showing axoneme formation defects (Fig 2H). Proper individualization complex organization was rescued by the transgenic maca-EGFP in the macanull/null background. We concluded that maca is required for proper individualization complex organization in the late stage of the sperm individualization process. Loss of proper sperm individualization explains the loss of mature sperm and the complete male sterility in macanull/null.

Kl-2, Kl-3, and Kl-5 protein levels are reduced in macanull/null testes To start to understand the molecular mechanism by which the sperm individualization complexes are impaired in macanull/null testes, we examined protein levels in a proteome-wide manner in four biological replicates each of the testes of maca+/+, macanull/+, macanull/null, and macanull/null with maca-EGFP rescue transgene, using mass spec with TMT-labeling (see Materials and Methods). Among the 6,559 proteins we detected, two proteins were significantly upregulated (relative abundance >1.3, adjusted P-value <0.05) and 33 proteins were significantly downregulated (relative abundance <0.77, adjusted P-value <0.05) in macanull/null compared with all the other three genotypes consistently (Figs 3 and S4A). Among these consistently dysregulated proteins, male fertility factor Kl-2 showed the strongest and most significant downregulation in macanull/null (relative abundance 0.12 and adjusted P-value 1.7 x 10−5 in macanull/null compared with macanull/+) (Figs 3 and S4A). Interestingly, two other male fertility factors Kl-3 and Kl-5 were also among the 33 consistently downregulated proteins. Kl-2 is an inner dynein arm (IDA) dynein heavy chain protein and Kl-3 and Kl-5 are outer dynein arm (ODA) dynein heavy chain proteins [23–28]. The male fertility factor genes kl-2, kl-3, and kl-5 encoding these proteins are all located in the Y chromosome long arm and span several megabases containing gigantic introns rich in repetitive sequences (Fig 1B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Proteins dysregulated in macanull/null testes including downregulated Kl-2, Kl-3, and Kl-5 revealed by mass spec. Heatmap of protein levels that were significantly dysregulated in the macanull/null testes compared with the testes from maca+/+, macanull/+, and macanull/null with maca-EGFP rescue transgene determined by mass spec with TMT labeling. Log 2 (fold-change compared with the means of macanull/+) are shown. Four biological replicates for each genotype. Two proteins (at the top) were significantly upregulated (relative abundance >1.3, adjusted P-value <0.05) and 33 proteins were significantly downregulated (relative abundance <0.77, adjusted P-value <0.05) in macanull/null compared with all the other three genotypes. Male fertility factor Kl-2 was most strongly and significantly downregulated (relative abundance 0.12 and adjusted P-value 1.7 x 10−5 in macanull/null compared with macanull/+). Proteins are sorted based on the chromosomal positions of the corresponding genes. https://doi.org/10.1371/journal.pgen.1009655.g003

No significant change in small RNA levels in macanull/null testes Next, we examined testis small RNAs (miRNAs, siRNA, and piRNAs) from three biological replicates each of maca+/+, macanull/+, macanull/null, macanull/+ with maca-EGFP rescue transgene, and macanull/null with maca-EGFP rescue transgene in a transcriptome-wide manner using high throughput sequencing. No miRNAs, siRNA, or piRNAs (including Y-linked Suppressor of Stellate (Su(Ste) piRNAs, which are crucial for male fertility[38]) showed significantly changed levels in macanull/null testes compared with the other four genotypes (S5 Fig).

kl-3 exon 13 skipping occurs in macanull/null testes We wondered if splicing dysregulation occurs in any transcripts in macanull/null testes. To test this, we analyzed our poly-A+ RNA-seq data using LeafCutter [39]. Curiously, we found that kl-3 mRNA exon 13 skipping occurs in macanull/null testes, but not in the other four genotypes (Figs 6B and S7). No other mRNA showed consistent splicing dysregulation in macanull/null compared with the other four genotypes. We also found that read mapping count was reduced rather evenly along the kl-2 and kl-5 gene regions in macanull/null compared with the other genotypes (Figs 6A, 6C, S6 and S8). In contrast, read mapping was reduced more severely in the kl-3 exon 13 region than in the other kl-3 coding regions in macanull/null, which is consistent with the exon 13 skipping (Figs 6B and S7). Thus, our poly-A+ RNA-seq data revealed kl-3 exon 13 skipping in macanull/null testes. No major mRNA level reduction, exon skipping, or protein level reduction for ks-1 (ory) was observed in macanull/null compared with the controls (S9 Fig).

maca mutation phenocopies kl-2, kl-3, and kl-5 RNAi knockdown Gene mutation or RNAi-mediated knockdown of kl-2, kl-3, or kl-5 in testis germline cells causes spermatogenesis defects including a scattering of the individualization complexes and male sterility [27,29,39]. We first confirmed that kl-2, kl-3, or kl-5 RNAi knockdown depleted corresponding cytoplasmic mature mRNAs, but not the nuclear precursor mRNA (pre-mRNA) transcripts in spermatocytes (S10 Fig). kl-2, kl-3, or kl-5 RNAi knockdown caused loss of mature motile sperm in seminal vesicles (S11A Fig) and scattering of the individualization complexes in the late stage of the sperm individualization process (S11B and S11C Fig), similar to maca mutation (Fig 2C, 2G and 2H). Thus, maca mutation and kl-2, kl-3, or kl-5 RNAi knockdown depleting their cytoplasmic mRNAs phenocopy each other.

kl-2 transcript level reduction in macanull/null testes detected with RT-qPCR To validate the strong kl-2 mRNA level reduction observed in poly-A+ RNA-seq (Figs 4B, 5B, S4B and S6), we quantitated kl-2 transcripts in testis using RT-qPCR. We performed the reverse transcription (RT) step using random primers so that precursor transcripts including transcription intermediates before poly-A addition as well as mature poly-A+ mRNAs can be detected in the RT-qPCR assays. When qPCR primer set that amplifies a region in kl-2 exon 1 was used, macanull/null showed a dramatic reduction compared with the control macanull/+ (became 0.07 fold) (Fig 7A). Similarly, when qPCR primer sets spanning large kl-2 introns and that spanning normal size kl-2 introns were used, macanull/null showed a severe reduction (became 0.01–0.05 fold). Mature kl-2 mRNAs can be detected by all of these primer sets. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Quantification of kl-2 transcripts by RT-qPCR show their reduction in macanull/null testes. Quantification of kl-2 transcripts in testis RNA by RT-qPCR. The RT step was performed with random hexamer primers. (A) qPCR amplicons targeted by designed primer sets are indicated by orange bars. The primers span exon-exon junctions to amplify only spliced transcripts, except for the first amplicon, which targets exon 1. Black lines indicate intron regions not included in the amplicons. (B) qPCR amplicons targeted by designed primer sets are indicated by gray and orange bars, which indicate intron and exons regions, respectively. At least one primer in each primer set targets an intron and therefore the primer sets target kl-2 transcripts before splicing (pre-mRNAs). Data were normalized to actin5C mRNA and the means of macanull/+. Mean +/- SD (n = 3 biological replicates). P-values <0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *. https://doi.org/10.1371/journal.pgen.1009655.g007 Next, we used qPCR primer sets to amplify regions in kl-2 introns and regions located at kl-2 exon-intron or intron-exon boundaries. kl-2 pre-mRNA transcripts before splicing, but not mature kl-2 mRNAs after splicing, can be detected by these qPCR primer sets. With these primer sets, the reduction degree in macanull/null compared with the control became gradually stronger as a primer set target location is shifted from the 5′ end to the 3′ end of the kl-2 gene (Fig 7B). Yet, the reduction degrees in macanull/null detected using these primer sets targeting kl-2 pre-mRNAs (Fig 7B) were not as strong as those obtained using the primer sets targeting the kl-2 exon regions (Fig 7A). The kl-2 transcript level was rescued in macanull/null with maca-EGFP rescue transgene in most cases (Fig 7A and 7B). Thus, in macanull/null testes, the kl-2 pre-mRNA transcript levels are more strongly reduced toward the 3′ end and the mature kl-2 mRNA levels are dramatically reduced, suggesting that later steps in the kl-2 mRNA transcription/processing/stability is more strongly affected than early steps.

Severe reduction of cytoplasmic mature kl-2 mRNA in macanull/null spermatocytes revealed by RNA FISH Next, we performed RNA in-situ hybridization (FISH) to visualize a spatiotemporal expression pattern of kl-2 transcripts in testes. In the control macanull/+ testes, we found that transcription of the kl-2 gene progresses in a highly spatiotemporal manner as previously shown for the kl-3 and kl-5 genes [40]. kl-3 and kl-5 expression during spermatocyte growth was subdivided into four stages based on their transcription progress from the 5′ end to the 3′ end of the genes [40]. Here we subdivided kl-2 expression in four stages as well. The transcription initiation occurs in early spermatocytes where neither kl-2 introns 1–2 nor exon 8 was detectable yet in the nucleus (stage 1) (Fig 8A and 8B). In the next stage, kl-2 introns 1–2 were expressed (stage 2). Then, kl-2 exon 8 was additionally expressed (stage 3). Finally, while the intron and exon signals were continued to be detected in the nucleus, mature kl-2 mRNAs that contain only exons, but not introns, were exported to the cytoplasm and reside as cytoplasmic kl-granules in late spermatocytes (stage 4). Thus, the gigantic kl-2 gene is transcribed gradually during the ~90-hour spermatocyte growth, and only in the late stage, kl-2 gene transcription is completed and spliced mature kl-2 mRNAs are exported to the cytoplasm. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. RNA FISH for kl-2 transcripts shows their reduction in macanull/null testes. RNA FISH to visualize kl-2 transcripts expression in (A, B) macanull/+ and (C, D) macanull/null testes. Introns 1–2 (green), exon 8 (red), and DAPI (white). (A, C) Apical regions of testes including the spermatocyte growth region are shown. The apical tip of testis is marked by *. Scale bars are 50 μm. (B, D) Single spermatocyte nuclei (white dashed line) at each stage of kl-2. Cytoplasmic mRNA granules are indicated by cyan arrows. Scale bars are 5 μm. https://doi.org/10.1371/journal.pgen.1009655.g008 In macanull/null testes, FISH signal for kl-2 pre-mRNA transcripts in the spermatocyte nucleus was weaker than in macanull/+ testes (Fig 8C and 8D). FISH signal for kl-2 exon 8 seemed more severely reduced than those for kl-2 introns 1–2. Furthermore, cytoplasmic mature kl-2 mRNA granule in stage 4 spermatocyte was almost completely absent in macanull/null testes. We concluded that kl-2 pre-mRNA transcript levels are detectably and cytoplasmic kl-2 mature mRNA levels are severely reduced in macanull/null testes, consistent with our RT-qPCR results (Fig 7).

kl-3 mRNA reduction and kl-3 exon 13 skipping in macanull/null testes confirmed by RT-PCR To validate the kl-3 mRNA level reduction and the kl-3 exon 13 skipping observed in the poly-A+ RNA-seq analysis (Figs 4B, 5B, 6B, S4B and S7), we performed RT-qPCR to measure kl-3 mRNA levels in testes. Primer sets spanning kl-3 introns to amplify mature kl-3 mRNAs revealed a reduction to 0.27–0.53 fold in macanull/null testes compared with macanull/+, except for one primer set (Fig 9A). The exception was the qPCR primer set with a forward primer targeting a region in exon 13 and a reverse primer targeting the junction between exon 13 and exon 14 (= spanning intron 13); it showed a much stronger reduction in macanull/null testes compared with macanull/+ (became 0.04 fold). The kl-3 mRNA levels were rescued in macanull/null with maca-EGFP rescue transgene. These results confirm the kl-3 mRNA level reduction and the kl-3 exon 13 skipping, consistent with the poly-A+ RNA-seq results. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 9. kl-3 mRNA reduction and exon 13 skipping in macanull/null testes revealed by RT-(q)PCR. (A) Quantification of kl-3 transcripts in testis RNA by RT-qPCR. The RT step was performed with random hexamer primers. qPCR amplicons targeted by designed primer sets are indicated by orange bars. The primers span exon-exon junctions to amplify only spliced transcripts. Black lines indicate intron regions not included in the amplicons. Data were normalized to actin5C mRNA and the mean of macanull/+. Mean +/- SD (n = 3 biological replicates). P-values <0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *. (B) Agarose gel electrophoresis of RT-PCR products using the indicated primers to examine kl-3 exon 13 skipping in testis RNA. (C) Diagram of Kl-3 protein domains. Amino acid residues 2866–3351 corresponding to the exon 13 is indicated with a magenta dash-lined yellow box. (D) Abundance of peptide fragments derived from Kl-3 protein determined by mass spec with TMT labeling plotted along the Kl-3 amino acid residue numbers. Data were normalized to the means of maca+/+. Mean +/- SD (n = 4 biological replicates). The amino acid residues 2866–3351 corresponding to the kl-3 mRNA exon 13 is indicated with a magenta dash-lined yellow box. (E) Abundance of Kl-3 protein determined using peptide fragments data (left) from the 1–2865 or 3352–4593 aa region or (right) from the 2866–3351 aa region, of Kl-3 in mass spec with TMT labeling. Data were normalized to the means of maca+/+. Mean +/- SD (n = 4 biological replicates). P-values <0.05 (Student’s t-test, unpaired, two-tailed) are indicated by *. (F, G) Western blots of testis lysates for Kl-3-3xFLAG using (F) a standard protocol and (G) an extended SDS-PAGE gel electrophoresis time to achieve better separation. The FLAG tag was inserted at the C-terminal end of Kl-3 at the endogenous kl-3 locus on the Y-chromosome [40]. https://doi.org/10.1371/journal.pgen.1009655.g009 To further confirm the kl-3 exon 13 skipping, we performed RT-PCR using testis RNA with a forward PCR primer targeting a region in exon 12 and a reverse PCR primer targeting a region in exon 14 and ran the PCR products on an agarose gel. A ~2.1 kb band showing exon 13 retention was predominantly obtained from the testis RNA of the controls (maca+/+ and macanull/+) and macanull/null with maca-EGFP rescue transgene, while a ~0.6 kb band showing exon 13 skipping was predominantly obtained from the testis RNA of macanull/null (Fig 9B). These results confirmed the kl-3 exon 13 skipping in macanull/null testes. Similarly, we detected the kl-3 exon 13 skipping when whole adult male RNA was used for RT-PCR analysis (S12A Fig). The ~2.1 kb PCR product showing kl-3 exon 13 retention was obtained from maca+/+ and macanull/+ males while the ~0.6 kb PCR product showing exon 13 skipping was obtained from macanull/null males. Consistent with the exclusive expression of maca in testes (Fig 1D and 1E), no PCR bands were obtained when whole adult female RNA was used (S12A Fig). In contrast, sex-specific alternative splicing of sxl, tra, and msl2, was not dysregulated in macanull/null males and females compared with maca+/+ and macanull/+ (S12B–S12D Fig). These results demonstrated that splicing dysregulation in macanull/null is specific to kl-3 exon 13 skipping, consistent with the poly-A+ RNA-seq analysis.

kl-3 exon 13 skipping causes an internal deletion of Kl-3 protein in macanull/null testes The kl-3 mRNA exon 13 skipping would result in an internal deletion of amino acid residues 2,866–3,351 of Kl-3 protein without causing a translation frameshift (Fig 9C). We wondered if the internally deleted Kl-3 protein is expressed in macanull/null testes. To test this, we first searched our TMT-labeled testis-proteome mass spec data for peptide fragments containing the 2,865–2,866 residues or the 3,351–3,352 residues of the full-length Kl-3 protein, which would derive from the full-length Kl-3 protein produced from kl-3 mRNA with the exon 13-retained, or for peptide fragments containing the residue 2,865 followed by the residue 3,352, which would derive from the internally deleted Kl-3 protein variant produced from the exon 13-skipped mRNA. However, none of these fragments was detected in our data, which has ~20% coverage for Kl-3 protein. Next, we took an alternative approach. The aa residues 1–2,865 and 3,352–4,593 are common between the full-length Kl-3 and the internally-deleted Kl-3 proteins while the aa residues 2,866–3,351 are specific to the full-length Kl-3. We analyzed the relative abundance of detected Kl-3 peptide fragments along the full-length Kl-3 amino acid sequence. Relative abundances of peptide fragment levels derived from the 2,866–3,351 amino acid residues were more strongly reduced in macanull/null compared with the peptide fragment levels derived from the other Kl-3 region (Fig 9D). The Kl-3 protein level was calculated to be 0.46 fold in macanull/null testes compared in maca+/+ when the peptide fragment data derived from the aa residues 1–2,865 and 3,352–4,593 were analyzed, while it was 0.19 fold when the peptide fragment data derived from the aa residues 2,866–3,351 were analyzed (Fig 9E). These mass spec data supported that kl-3 exon 13 skipping causes an expression of the internally deleted Kl-3 protein variant in macanull/null testes. Furthermore, we attempted to detect the internally deleted Kl-3 protein variant using Western blot. We utilized a C-terminally 3xFLAG tagged kl-3 gene at its endogenous locus in the Y-chromosome [40]. While the size difference of the C-terminally 3xFLAG tagged Kl-3 protein between in macanull/+ and in macanull/null was not apparent in a standard protocol Western blot (Fig 9F), the size difference became apparent when gel electrophoresis time was much extended to achieve better separation (Fig 9G). The detected Kl-3 protein size was smaller in macanull/null than in macanull/+, supporting the idea that the full-length protein is predominantly expressed in macanull/+ testes while the internally deleted Kl-3 protein is predominant in macanull/null testes. When testis protein lysate from macanull/+ and that from macanull/null were mixed and loaded together on a Western blot gel, it produced double bands, confirming the existence of two different size Kl-3 proteins (Fig 9G). Taken together, we concluded that the kl-3 mRNA exon 13 skipping in macanull/null testes causes expression of the Kl-3 protein variant with an internal deletion of the corresponding amino acid residues. The deleted region contains the entire Dynein heavy chain AAA module D4 domain and the N-terminal half of the Dynein heavy chain coiled coil stalk domain of the full-length Kl-3 (Fig 9C). The Kl-3 protein activity is likely to be significantly impaired by this deletion.

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009655

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.