Drosophila YBX1 homolog YPS promotes ovarian germ line stem cell development by preferentially recognizing 5-methylcytosine RNAs

5-Methylcytosine (m5C) is a RNA modification that exists in tRNAs and rRNAs and was recently found in mRNAs. Although it has been suggested to regulate diverse biological functions, whether m5C RNA modification influences adult stem cell development remains undetermined. In this study, we show that Ypsilon schachtel (YPS), a homolog of human Y box binding protein 1 (YBX1), promotes germ line stem cell (GSC) maintenance, proliferation, and differentiation in the Drosophila ovary by preferentially binding to m5C-containing RNAs. YPS is genetically demonstrated to function intrinsically for GSC maintenance, proliferation, and progeny differentiation in the Drosophila ovary, and human YBX1 can functionally replace YPS to support normal GSC development. Highly conserved cold-shock domains (CSDs) of YPS and YBX1 preferentially bind to m5C RNA in vitro. Moreover, YPS also preferentially binds to m5C-containing RNAs, including mRNAs, in germ cells. The crystal structure of the YBX1 CSD-RNA complex reveals that both hydrophobic stacking and hydrogen bonds are critical for m5C binding. Overexpression of RNA-binding–defective YPS and YBX1 proteins disrupts GSC development. Taken together, our findings show that m5C RNA modification plays an important role in adult stem cell development.

Fly LMBR1/LIMR-type protein Lilipod promotes germ-line stem cell self-renewal by enhancing BMP signaling

Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. However, their biological function is unknown. Here, we show that the fly family member Lilipod (Lili) is required for germ-line stem cell (GSC) self-renewal in the Drosophila ovary where it enhances bone morphogenetic protein (BMP) signaling. lili mutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factor bam. Conversely, overexpressed Lili induces supernumerary pMad-positive bamP-GFP–negative GSCs. Interestingly, differentiation of lili mutant GSCs is bam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing a bam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first, to our knowledge, in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche.

144. MAELSTROM - A PROTEIN THAT IS ESSENTIAL FOR SPERMATOGENESIS AND TRANSPOSABLE REPRESSION IS EXPRESSED IN ADULT OVARY OF MAMMALS AND BIRDS

Maelstrom (MAEL) is a highly evolutionarily conserved protein located at the perinuclear structure of animal germ cells called nuage. The MAEL protein contains HMG and Tudor domains and associates with components of the piRNA and RNAi pathways and chromatin remodelling factors. Recent work has shown that MAEL is required for the differentiation of the germ-line stem cell lineage and for the retroposon repression. In mouse, Mael is expressed in male germ cells and is essential for spermatogenesis and retroposon suppression. We have investigated the evolution of the Mael gene in mammals and birds. As expected the gene is highly conserved in all three mammalian lineages and in chicken. Interestingly, the platypus MAEL has exclusive changes in the DnaQ-H 3’-5’ exonuclease domain and computational modelling suggested that these changes may affect the folding of the protein. Expression analysis revealed that the Mael gene is transcribed in testis but also in adult ovaries of chicken, platypus, mouse and human. In situ hybridisation of the Mael transcript on ovary sections of mouse and platypus shows that gene expression is found in pre-antral and antral follicles. The data so far also showed some differences in the expression pattern between mouse and platypus. In mouse, we detected transcript in oocyte, granulosa cells and cumulus cells whereas in the platypus we only observed expression in oocyte. Earlier work demonstrated that Drosophila Mael mutant ovaries had mislocalisation of the RNAi pathway proteins, Dicer and Argonaute2. It is well known that RNAi pathyway is involved in the repression of transposon in the testis and ovary across animal kingdom. As a key component of the RNAi pathway, MAEL is reported to co-localise and interact with MILI and MIWI proteins. These finding may suggest a role of MAEL in retroposon control in ovary and folliculogenesis.