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 heatshock-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. ARTICLE HISTORY

Background Differences in core or tissue‐specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22‐like. Unlike ubiquitously expressed eRpL22, eRpL22‐like expression is tissue‐specific, notably within the male germline and the eye. We investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. Results Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22‐like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22‐like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi‐mediated eRpL22‐like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22‐like across development, with the variant at the expected molecular mass co‐sedimenting with active ribosomes. Conclusion Our data reveal differential patterns of eRpL22‐like expression relative to eRpL22 and suggest a specific role for eRpL22‐like in developmental patterning of the eye.