Two RNA Binding Proteins, ADAD2 and RNF17, Interact to Form a Heterogeneous Population of Novel Meiotic Germ Cell Granules With Developmentally Dependent Organelle Association

Abstract

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells.

Introduction

The male germ cell relies on complex RNA biology for successful differentiation. As a result, they express and require a wide range of RNA binding proteins (RBPs), many of which are housed in non-membrane bound, cytoplasmic organelles termed germ cell RNA granules or germ cell granules. These granules are especially prevalent during meiotic and post-meiotic germ cell differentiation and are fundamental for proper developmental progression in multiple species [1,2]. In mammalian male germ cells for example, six types of granules have been identified via electron microscopy (EM), five of which can be found in meiotic spermatocytes [3]. Loss of core granule proteins commonly leads to meiotic or post-meiotic germ cell arrest [4–8], underscoring their importance in germ cell differentiation and male fertility.

Materials and method

Ethics statement

All animal use protocols were approved by the Rutgers University animal care and use committees. Mouse procedures were conducted according to relevant national and international guidelines as outlined in the Guide for the Care and Use of Laboratory Animals and provisions set forth by the Animal Welfare Act. Adherence to these guidelines was overseen on the institutional level by the Rutgers Institutional Animal Care and Use Committee and all animal procedures were approved under Rutgers IACUC ID TR202000034.

Results

ADAD2 interacts with RNF17, a testis-specific RNA binding protein
The ADAD2 granule appears to be distinct from the best characterized spermatocyte granule, intermitochondrial cement (IMC) [24]. To better determine the molecular nature of the ADAD2 granule, we immunoprecipitated ADAD2 from wildtype (n = 3) and Adad2 mutant (Adad2 M/M, n = 1) testes at 42 days post-partum (dpp) followed by mass spectrometry (IP-MS) to identify potential ADAD2-granule associated proteins (Fig 1A and S1 Table). Hits identified in all wildtype samples but not the mutant included well characterized post-meiotic germ cell proteins along with several RNA binding proteins (Fig 1B). Of the significant peptides identified in wildtype only, ADAD2-derived peptides represented nearly a tenth, confirming efficacy of pulldown. However, the highest number of peptides identified belong to another RNA binding protein, ring finger protein 17 (RNF17). RNF17 peptides comprised over a fifth of those identified as significant. RNF17, like ADAD2, is testis-specific and has been reported to form a spermatocyte granule [8].

Discussion

In mammalian meiotic male germ cells, germ cell granules are important sites of RNA metabolism required for their normal differentiation. Traditionally, spermatocytes are thought to contain five morphologically distinct granule types and loss of many granule components leads to male infertility. In spite of this, there is limited knowledge regarding the protein composition of, function of, and relationship between these granules. To that end, this work aimed to characterize a recently identified granule that contains the RNA binding protein ADAD2, which is required for male fertility [24]. After identifying a second RNA binding protein, RNF17 [8], as an ADAD2 interacting partner, genetic knockout models combined with localization studies demonstrated ADAD2 and RNF17 are co-dependent on one another to form multiple distinct populations of ADAD2-RNF17 granules. 

Acknowledgments

The authors would like to thank current and previous members of the Snyder laboratory including Yeva Shamailova, Aditi Badrinath, Kelly Seltzer, Christopher Eddy, Gabriella Acoury, Gabrielle Vittor, and Megan Forrest for their support with animal husbandry, molecular analyses, and critical evaluation throughout the project. We would additionally like to thank Drs. Jeremy Wang and Xin Li for the kind gift of Rnf17 mutant mice, the Human Genetics Institute of New Jersey Imaging Core for their support in confocal imaging, and Dr. Jessica Shivas and Daniel Jung for their assistance with confocal data acquisition and analyses.

Citation: Chukrallah LG, Potgieter S, Chueh L, Snyder EM (2023) Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association. PLoS Genet 19(7): e1010519. https://doi.org/10.1371/journal.pgen.1010519

Editor: Wei Yan, The Lundquist Institute, UNITED STATES

Received: November 10, 2022; Accepted: June 17, 2023; Published: July 10, 2023

Copyright: © 2023 Chukrallah et al. 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.

Data Availability: All data are in the manuscript and/or supporting information files.

Funding: The work described herein was supported by financial support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIH-NICHD F32 HD072628, K99/R00 HD083521, and R01 HD107066 to EMS) and Rutgers University (to EMS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have no competing interests to declare.