The silkworm W chromosome is a source of female-enriched piRNAs

Abstract

In the silkworm, Bombyx mori, the W chromosome plays a dominant role in female determination. However, neither protein-coding genes nor transcripts have so far been isolated from the W chromosome. Instead, a large amount of functional transposable elements and their remnants are accumulated on the W chromosome. PIWI-interacting RNAs (piRNAs) are 23-30-nt-long small RNAs that potentially act as sequence-specific guides for PIWI proteins to silence transposon activity in animal gonads. In this study, by comparing ovary- and testis-derived piRNAs, we identified numerous female-enriched piRNAs. Our data indicated that female-enriched piRNAs are derived from the W chromosome. Moreover, comparative analyses on piRNA profiles from a series of W chromosome mutant strains revealed a striking enrichment of a specific set of transposon-derived piRNAs in the putative sex-determining region. Collectively, we revealed the nature of the silkworm W chromosome as a source of piRNAs.

FIGURE 1.

piRNA sex dimorphism in the silkworm. (A) Log₂ fold ratio between normalized ovarian and testis-derived piRNA reads against 121 well-annotated transposons. (Red bars) Transposons showing higher content of ovarian piRNAs [WT(OV)/WT(TE) > 2]. (Blue bars) Transposon showing WT(TE)/WT(OV) > 2. (B) Density plots for representative transposons that show sex-biased piRNA content. Ovarian piRNAs (red); testis-derived piRNAs (blue). Normalized reads are indicated in reads per million (RPM).

FIGURE 2.

The W chromosome is a source of female-enriched piRNAs. (A) Chromosomal distribution of indicated transposons. We mapped indicated transposons to the B. mori genome and W-BAC sequences using BLAST. Color intensity indicates the maximum length of the mapped transposon (E-value < 1 × 10⁻⁵⁰). (B) Single nucleotide polymorphism between W chromosome-derived piRNA (W-type) and autosomal piRNA (A-type). Expression levels (RPM) of each type of piRNA in ovarian and testis-derived libraries are shown.

FIGURE 3.

Transposons and associated piRNAs derived from the sex-determining region. (A) Strains with several types of W chromosomes. LY's W chromosome lacks 11 of 12 known W chromosome-specific RAPD markers, but it retains the sex-determining ability. MW is an introgression line that harbors W chromosome of B. mandarina (see text), which can determine the sex of B. mori. The W chromosome fragment attached to the Z chromosome cannot determine femaleness. (B) Transposons enriched in the sex-determining region. The heat map indicates relative expression of piRNAs from five libraries matching each transposon (Supplemental Table S4). (C) Representative transposons enriched in the sex-determining region (SDR).

FIGURE 4.

The nature of the silkworm W chromosome as a source of female-enriched piRNAs. The silkworm W chromosome contributes to production of female-enriched piRNAs. Primary piRNAs can be produced from defective transposon loci in the W chromosome and autosomes. Female-enriched secondary piRNAs can be generated through cleavage of W chromosome-derived sense transposon mRNAs by such primary piRNAs. Female-enriched piRNAs such as Pakurin-derived piRNAs can be produced both from SDR and non-SDR loci in the W chromosome. A set of female-enriched piRNAs such as Judo-derived piRNAs shows a striking enrichment in the SDR.