Functional interplay between ribosomal protein paralogues in the eRpL22 family in Drosophila melanogaster

Abstract

Duplicated ribosomal protein (RP) genes in the Drosophila melanogaster eRpL22 family encode structurally-divergent and differentially-expressed rRNA-binding RPs. eRpL22 is expressed ubiquitously and eRpL22-like expression is tissue-restricted with highest levels in the adult male germline. We explored paralogue functional equivalence using the GAL4-UAS system for paralogue knockdown or overexpression and a conditional eRpL22-like knockout in a heat- shock flippase/FRT line. Ubiquitous eRpL22 knockdown with Actin-GAL4 resulted in embryonic lethality, confirming eRpL22 essentiality. eRpL22-like knockdown (60%) was insufficient to cause lethality; yet, conditional eRpL22-like knockout at one hour following egg deposition caused lethality within each developmental stage. Therefore, each paralogue is essential. Variation in timing of heat-shock-induced eRpL22-like knockout highlighted early embryogenesis as the critical period where eRpL22-like expression (not compensated for by eRpL22) is required for normal development of several organ systems, including testis development and subsequent sperm production. To determine if eRpL22-like can substitute for eRpL22, we used Actin-GAL4 for ubiquitous eRpL22 knockdown and eRpL22-like-FLAG (or FLAG-eRpL22: control) overexpression. Emergence of adults demonstrated that ubiquitous eRpL22-like-FLAG or FLAG-eRpL22 expression eliminates embryonic lethality resulting from eRpL22 depletion. Adults rescued by eRpL22-like-FLAG (but not by FLAG-eRpL22) overexpression had reduced fertility and longevity. We conclude that eRpL22 paralogue roles are not completely interchangeable and include functionally-diverse roles in development and spermatogenesis. Testis-specific paralogue knockdown revealed molecular phenotypes, including increases in eRpL22 protein and mRNA levels following eRpL22-like depletion, implicating a negative crosstalk mechanism regulating eRpL22 expression. Paralogue depletion unmasked mechanisms, yet to be defined that impact paralogue co-expression within germ cells.

Keywords: Cell biology; RNAi; fertilization; gene regulation; molecular genetics; reproduction.

Figure 1.

eRpL22 family paralogue knockdown generates differential outcomes in fly development. A & A’) F1 progeny were scored to assess the impact of knockdown (KD) of eRpL22 paralogues on development. The lack of F1 adults harboring Actin-GAL4 and eRpL22.IR, represented as unbalanced progeny (lacking CyO and TM3 balancers), suggests eRpL22 is an essential gene (a). Equal representation of the CyO balancer and Actin-GAL4 driver, with no significant differences in progeny number or sex ratios compared to Act>GFP control (left), suggests eRpL22-like is dispensable for viability (right) (a’). F: Females; M: Males. Numbers of progeny scored are indicated below each genotype. b) Western analysis of testis tissue confirms eRpL22-like knockdown (Act>eRpL22-like.IR). Tubulin was used as a loading control. c) Western analysis of testis tissue confirms an 80% eRpL22-like knockdown (bam>eRpL22-like.IR). Tubulin was used as a loading control.

Figure 2.

Conditional, heat shock-induced knockout of eRpL22-like. a) Diagram of the eRpL22-like locus (top) and diagram of the FRT flanked eRpL22-like locus before (middle) and after heat shock induced FLP expression (bottom). b) PCR confirmation of eRpL22-like knockout showing an expected shift in PCR products corresponding to FLP-FRT mediated excision of genomic sequence. c) Dissecting scope images show gross morphological consequences of eRpL22-like knockout on larval development. Differences in overall larval growth are apparent. White arrows how differences in trachea development with and without heat shock. Scale bar: 400 μm, red box scale bar: 100 μm. d) IHC analysis of Malpighian tubules (white arrow) at day 5 post embryo deposition. Tissue is stained against eRpL22-like (Green) and DAPI (Blue) is used as a nuclear stain. Scale bar: 20 μm. Heat shock treatment shows no effect on eRpL22-like expression and localization. CKOFLP tissue does not recapitulate wild type eRpL22-like staining, indicating potential differences in eRpL22-like expression between wild type and CKOFLP. Heat shock treatment results in dramatic defects in Malpighian tubule development. e) Diameter of Malpighian tubes was quantified using ImageJ. Malpighian tubules are significantly smaller. Error bars represent one standard deviation, *p < 0.01 according to t-test. f) Overall fluorescence was measured and normalized to the number of cells within the area measured to control for growth differences. The number of cells was determined by counting nuclei of each tube.

Figure 3.

Effect of eRpL22-like knockout on testes development. a) Dissecting microscope images of gross morphological changes to adult testis development resulting from heat shock treatment of embryos at 24–36 hours post-embryo deposition ([HPED]; bottom left) and 48–60 HPED (bottom right). Heat shock 24–36 HPED results in severe defects in both testis and accessory gland growth. Heat shock 48–60 HPED results in overall normal reproductive system. (+, CKO; +, CKO; HS-FLP/+). Scale bar: 300 μm. T- testis, AG- accessory gland, SV- seminal vesicle. SV/T indicates the tissue in the labeled region is either malformed testis, seminal vesicle, or both tissues. b) eRpL22-like knockout was confirmed through PCR analysis of DNA extracted from heat-shock treated CKO; HS-FLP/+ testis tissue compared to untreated control tissue.

Figure 4.

Assessment of fertility in paralogue knockdown and rescue genotypes determined by number of eclosed progeny. F1 male fertility was assessed by number of eclosed progeny per male when mated with a single wildtype female. Compared to all other genotypes, eRpL22-like-FLAG rescue males have a significant decrease in number of progeny per male. eRpL22-like paralogue knockdown was performed with bam-GAL4-VP16. eRpL22-like-FLAG overexpression was driven by Actin-GAL4. The eRpL22-like OE and eRpL22 OE rescues were driven with Actin-GAL4. FLAG (FL), knockdown (KD), overexpression (OE), and rescue are listed below genotypes. Error bars represents one standard deviation, * represents a p < 0.01 according to t-test, graph represents the mean of 10 males. The genotypes of males from each fertility cross are listed from left to right: Wildtype, bam>eRpL22-like.IR, Act>eRpL22-like-FLAG, Act>eRpL22-like-FLAG/eRpL22.IR, Act>FLAG-eRpL22/eRpL22.IR.

Figure 5.

Rescue of embryonic viability is achieved by overexpression of eRpL22-like in flies ubiquitously depleted of eRpL22. (a) Western analysis shows no change in accumulation of eRpL22 or eRpL22-like levels in testes from 3 day old males. In gonadectomized bodies, eRpL22-like is detected due to ubiquitous overexpression (OE) in all tissues (using Act>eRpL22-like-FLAG). Western blot analysis shows a ~ 15% loss of unmodified eRpL22 (33kD) and a ~ 65 % loss of SUMOylated eRpL22 (~≥55kD). Tubulin was used as a loading control. (b) Representative phase contrast microscopy images of wildtype control and eRpL22-like-FLAG OE rescue testes from 2 day old males. All stages of spermatogenesis are evident; however, an increase in immature spermatocytes (brackets) is noticeable within eRpL22-like OE rescued testes. An asterisk denotes the apical tip. (10x magnification). (c) IHC of wildtype control or eRpL22-like OE rescued from testes of 3 day old males. End of spermatid bundles (arrow) are closer to the apical tip (asterisk), and degenerating cysts are present (arrowhead). (10x magnification) Images in B and C are representative of rescued testes. Control is age matched wildtype males.

Figure 6.

Mitotic germline eRpL22 paralogue depletion or overexpression affects opposing paralogue expression levels. a) An increase in testis eRpL22 protein levels is observed when eRpL22-like is specifically depleted in the male germline (bam-GAL4-VP16) when compared to control (wildtype). The most notable increase occurs in the unmodified (33kD) species (arrow). Testis eRpL23a levels remain constant. b) qRT-PCR shows a statistically significant increase (average: 31% [1.31 fold change]; range: 17–47%) in testis eRpL22 mRNA levels when eRpL22-like is depleted from the germline. Error bars represent standard error, n = 3, *p < 0.01 according to t-test. c) eRpL22-like overexpression (using an Actin-GAL4 driver) results in a decrease of eRpL22 in the Drosophila testis and bodies compared to controls (wildtype). Significant knockdown is seen in the unmodified (33kD) species of eRpL22 (arrow). FLAG signal confirms the presence of the overexpression construct eRpL22-like-FLAG. Tubulin was used as loading control for testes samples and protein assays were used as a loading control for gonadectomized bodies. d) qRT-PCR shows a statistically significant increase (average: 311% [fold-change 4.11]) in gonadectomized bodies eRpL22-like mRNA levels when eRpL22-like is ubiquitously overexpressed. Error bars represent one standard deviation, n = 3, *p < 0.01 according to t-test. e) qRT-PCR shows a reduction (average: 44% [0.56 fold-change]) in eRpL22 mRNA levels in gonadectomized bodies from ubiquitous overexpression of eRpL22-like. Error bars represent one standard deviation, n = 3. f) qRT-PCR shows a significant reduction (average: 69% [0.31 fold-change]) in testis eRpL22 mRNA levels when eRpL22-like is ubiquitously overexpressed. Error bars represent one standard deviation, n = 9, *p < 0.01 according to t-test g) qRT-PCR shows a significant reduction (average: 53% [0.47 fold-change]) in testis eRpL22-like mRNA levels upon ubiquitous eRpL22-like overexpression. Error bars represent one standard deviation, n = 9, *p < 0.01 according to t-test.

Figure 7.

Summary of eRpL22 knockdown and rescue effects. Outcomes from eRpL22-depletion are shown in boxes. Arrows are color-coded (green: eRpL22-like; red: eRpL22) to depict paralogue gene overexpressed for rescue. Ubiquitous expression was driven by Actin-GAL4. KD: knockdown; OE: overexpression; FL: FLAG.