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

Abstract

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.

Keywords: GSC; RNA methylation; YBX; YPS; m5C.

Fig. 1.

YPS is required intrinsically to promote GSC maintenance, proliferation, and differentiation in the Drosophila ovary. (A) Schematic diagram of the germarium: spectrosome (red dot) and fusome (red line). TF, terminal filament; CPC, cap cells; IGS, inner germarial sheath cells; FC, follicle cell. Ovals and arrows indicate cap cells and spectrosomes in GSCs, respectively, whereas arrowheads and asterisks denote spectrosomes in CBs and fusomes in the cysts, respectively. (B–D) Control germarium carries three GSCs and no CBs (B), while yps mutant germaria contains two GSCs and four CBs (C). (D) Quantification results. (E–G) yps mutant germaria contain fewer EdU-positive GSCs than controls. G shows quantification results. Arrows in E indicate EdU⁺ GSCs; arrowhead in F indicates EdU⁺ CB. (H) mRNA in situ hybridization results show that yps mRNA (green) is expressed primarily in GSCs, CBs, and cysts. (I–L) Germ line-specific, but not niche-specific, knockdown significantly decreases GSCs and increases CBs. L presents quantification results. (M–P) Ubiquitous expression of human Ybx1 can rescue the defects in GSC maintenance, proliferation, and differentiation in the yps mutant.

Fig. 2.

Drosophila YPS preferentially bind to m⁵C-containing RNAs in germ cells. (A–H) Germ line-specific Flag-YPS and anti-m⁵C antibody pull down mostly overlapped total RNAs, rRNAs, tRNAs, LncRNAs, premiRNAs, asRNAs, snoRNAs, snRNAs, and mRNAs (numbers represent different RNAs). (I and J′) YPS-binding sites are closely correlated with m5C sites in mad and eIF4A mRNAs (I and I′) and in mei-P26 and sxl mRNAs (J and J″). (K and L) m⁵C sites and YPS-binding sites are more prevalent in the CDS and intron than in the 5′ UTR and the 3′ UTR. (M) Top 15 significantly overrepresented pathways for the mRNAs sharing YPS-binding sites and m5C sites based on KEGG pathway analysis.

Fig. 3.

The CSDs of Drosophila YPS and human YBX1 preferentially bind to the m⁵C-containing RNA. (A and B) ITC analysis of Drosophila YPS binding to m⁵C-containing and RNA oligos. (C and D) ITC analysis of human YBX1 binding to m⁵C-containing and RNA oligos. (E and F) Overall structure of the human YBX1 CSD domain in complex with to m⁵C-containing and RNA oligos. The m⁵C RNA is shown in sticks, while the YBX1 CSD is shown in cartoon. The aromatic residues that interact with the RNA bases are indicated. (G) Detailed presentation of the interactions between YBX1 CSD domain aromatic residues and individual m⁵C RNA bases. The residues in the CSD domain and the m⁵C RNA bases are shown in cyan and orange, respectively.

Fig. 4.

Germ line-specific expression of RNA binding-defective yps and Ybx1 mutants severely compromises GSC development. Ovals indicate cap cells, whereas arrows and arrowheads point to spectrosomes in GSCs and CBs, respectively. (A–F′) Germ line-specific expression of yps^W68A, Ybx1^w65A, and Ybx1^F74A, but not of Ybx1^H87A, significantly decreases GSCs and increases CBs compared with overexpression of wild-type yps and Ybx1. F and F′ show results. Some yps and Ybx1 mutant-expressing germaria contain no GSCs (C and E) or one GSC (D), whereas other germaria accumulate many CBs (C′ and D). (G–K) Germ line-specific expression of yps or Ybx1 mutants except Ybx1^H87A decreases EdU⁺ GSCs compared with controls. In G, the arrow indicates EdU⁺ GSC. K shows quantification results.