Pivotal roles of the ribosome in proteome production and of extrinsic factors in regulating translation are well established. Relatively unexplored is how ribosome composition itself impacts specific mRNA translation during cellular differentiation or development. Assembly of ribosomal protein (RP) paralogues increases ribosome heterogeneity and the prospect of functional diversity in the ribosomal pool. Compelled by this, our long term goal is to understand how ribosome heterogeneity expands the regulatory capacity of the translation apparatus in differentiation. We have investigated eRpL22 paralogue expression in the Drosophila male germline as a model to test the hypothesis that heterogeneous ribosomes (distinguished by eRpL22 or eRpL22-like paralogue content) are functionally diverse ? translating unique subpopulations of mRNAs on each ribosome type during sperm differentiation. Indeed our recent RNAseq data (compiled from polysomes immunoprecipitated with paralogue-specific antibodies) show that ~50% of adult testis mRNAs are primarily enriched on one or the other ribosome type by an unknown selection mechanism. Identification of mRNAs known to be expressed in mitotic spermatogonia confirms that both paralogues function as ribosomal components within this mitotic cell lineage, with constitutively expressed mRNAs enriched on eRpL22-polysomes and most mitotic mRNAs distributed on both types. A role for eRpL22-like- polysomes in translating most meiotic and post-meiotic stage mRNAs is further supported. We have also shown that eRpL22 accumulation in the ribosomal pool is regulated, at least in part, by testis-specific post- translational modifications and nucleoplasmic sequestration within meiotic spermatocyte nuclei, as well as by negative control of eRpL22 levels by eRpL22-like through an unknown RNA-mediated mechanism. That a pool of eRpL22-ribosomes persists after meiosis is supported by enrichment of a subset of post-meiotic cup mRNAs on eRpL22-polysomes. Informed by preliminary data and using a combination of molecular, cellular, biochemical, computational, and genetic approaches to focus on mechanisms involved in directing mRNAs to specific ribosome types during sperm development, we propose: (1) to determine (in transfected S2 lines expressing eRpL22-like) if preferential entry of specific mRNAs (identified by RNAseq) onto specific ribosome types requires germ cell-specific interactors or if intrinsic features of ribosomes are sufficient to direct entry and (2) to determine the ribosomal and functional status of both paralogues in meiotic spermatocytes and mature sperm, and to quantify levels of testis-specific mRNAs isolated from eRpL22- or eRpL22-like ribosomes from meiotic arrest mutants and a fertility-deficient eRpL22 knockdown/ eRpL22-like overexpression transgenic fly line. New principles should emerge that refine our understanding of a regulatory role for specialized ribosomes in recruiting mRNAs for translation specificity in spermatogenesis.

Public Health Relevance

Developmental processes that lead to the production of mature, fully fertile sperm are conserved in Drosophila and mammalian systems and require systematic expression and proper regulation of a genetic program that transforms mitotic spermatogonia into mature sperm capable of fertilization. We have recently shown in the Drosophila male germline that heterogeneity in ribosome composition (distinguished by different ribosomal protein paralogue content) plays an important role in regulating translation of ~50% of testis mRNAs, where mRNAs are enriched on specific polysomes containing either eRpL22 or eRpL22-like. With implications for understanding factors regulating human sperm development and fertility, we will use a combination of molecular, biochemical, computational, cellular, and genetic approaches in the Drosophila system to investigate mechanisms by which specific mRNAs (some of which encode functions conserved for human sperm development) are targeted to specific ribosome types to produce sperm competent for fertilization.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM132889-01
Application #
9732152
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Reddy, Michael K
Project Start
2019-05-01
Project End
2022-04-30
Budget Start
2019-05-01
Budget End
2022-04-30
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Lehigh University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
808264444
City
Bethlehem
State
PA
Country
United States
Zip Code
18015