The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

Valeria Messina,Raffaele Geremia,Oliver Meikar,Noora Kotaja,Maria Paola Paronetto,Claudio Sette,Sara Calabretta,Emanuele Buratti

doi:10.1371/journal.pone.0039729

Abstract

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis.

Highlights

Spermatogenesis is characterized by a complex regulation of gene expression
As previously reported [20], SAM68 is localized in the nucleus of pachytene spermatocytes, it translocates into the cytoplasm of secondary spermatocytes and it localizes again in the nucleus of round spermatids (Figure 1A)
We observed that SAM68 accumulates in perinuclear, dense granules resembling the chromatoid body (CB) in secondary spermatocytes and early round spermatids (Figure 1A)

Summary

Introduction

Spermatogenesis is characterized by a complex regulation of gene expression. Male germ cells have to face an extended period of silencing of the genome, which occurs during meiotic homologous recombination and during morphological differentiation of haploid spermatids into spermatozoa [1]. Analysis of the protein content by mass spectrometry revealed that the bulk of the CB mass is composed of six proteins that are involved in different aspects of RNA processing. They include the RNA helicase DDX4/MVH (Mouse VASA Homologue), the PIWI protein named PIWIL1/ MIWI, the Tudor domain containing proteins TDRD6 and 7, the gonadotropin regulated testicular helicase DDX25/GRTH and the polyA binding protein PABPC3 [6]. The CB of round spermatids in Miwi, Grth, Tdrd and Tdrd knockout germ cells exhibits morphological abnormalities [8,9,10,11,12]. The CB seems to function as an RNA-processing centre in male germ cells, as previously demonstrated for the germ plasm of lower organisms [2]

Methods

Results

Conclusion