Association of germ granules and ribosomes in piRNA-mediated RNA regulation during spermatogenesis

Abstract

Male infertility due to various defects in spermatogenesis has been increasing rapidly for the past few decades. The successful completion of spermatogenesis requires strict post-transcriptional RNA regulation, and cytoplasmic germ cell-specific ribonucleoprotein granules, germ granules, are known to be important sites for this regulation. The purpose of this thesis was to examine the mechanisms of germ granule-associated RNA regulation pathways in male mouse germ cells, with a focus on the piRNA pathway. More specifically, the aim was to broaden the understanding of genic piRNA production, and its possible association with ribosomes and nonsense-mediated RNA decay pathway component SMG6. To reach this aim, the localization of gene-overlapping piRNA clusters in the genome was characterized visually, followed by characterization of the gene and piRNA expressions during the first wave of spermatogenesis using mRNA and small RNA sequencing. Eventually, the interaction of MIWI, SMG6 and ribosomes was studied using polysome fractionation of mouse testicular extracts. This study revealed that genic piRNA clusters align almost perfectly with the genes they overlap with, suggesting that all genic piRNAs are produced from mRNAs of the genes. Temporally, the expressions of piRNAs and the piRNA-hosting genes correlated only partially. SMG6, MIWI, piRNAs, and piRNA-producing mRNAs were all found associated with polysomes, suggesting it is possible that SMG6 affects the ribosome-associated production of piRNAs from mRNAs. However, the deletion of SMG6 didn’t affect the interaction of the other studied components, and thus, further research is needed to determine the function of SMG6 in piRNA production and to eventually understand the dynamics between germ granules and translation.