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 proteincoding 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.

A role for transcription from a piRNA cluster in de novo piRNA production

“…Small RNA fractions were extracted from the immunoprecipitates with the aid of a miRVana miRNA isolation kit (Ambion) according to the manufacturer's instruction. Reverse transcription and following qPCR were performed as described previously (Kawaoka et al 2011c). Primers used in this experiment were 59-TGAACTTCAG GGTCAGCTTGCCGTAGGT-39 for antisense GFP piRNA-1, 59-CTGAAGTTCATCTGCACCACCGGCAAGC-39 for sense GFP piRNA-1, and 59-TACTATACAACCTACTACCTCA-39 for let-7.…”

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein

“…The W chromosome is almost fully occupied by transposable elements or other repeat elements (3); therefore, it is very difficult for researchers to assemble long accurate contigs for this chromosome, which prevents the molecular identification of Fem. Given that transposons or other selfish elements are the precursors of PIWI-interacting RNAs (piRNAs), 2 we previously employed a piRNA sequencing-based approach to identify the transcripts produced from the B. mori W chromosome (4). Based on deep sequencing of piRNAs from the gonads of B. mori mutant strains, each of which possesses a unique W chromosome structure (5), we identified female-enriched piRNAs, which are produced from the putative sex-determining region of the W chromosome (4).…”

“…Among them, we found a single transcript that is expressed specifically in females throughout the embryonic stages. This transcript was produced from the W chromosome, and it was found to be a precursor of a female-specific piRNA using silkworm piRNA libraries (4,8,9). To determine the role of this female-specific piRNA, we designed an RNA inhibitor for this piRNA, injected it into female embryos, and examined the splicing patterns of B. mori doublesex (Bmdsx), a gene that acts at the end of the sex differentiation cascade (10,11).…”

The role of non-coding RNAs in male sex determination and differentiation

“…The silkworm Bombyx mori uses a ZW female: ZZ male sex chromosome system, with the W sex chromosome known to be female-determining. The female W chromosome is enriched in piRNAs, and one of these, dubbed Feminiser (Fem), acts as the primary female-sex determining gene (Kawaoka et al 2011, Kiuchi et al 2014. The Fem sequence is reiterated on the silkworm W sex chromosomes, and encodes a mature piRNA that is expressed in embryos and acts to regulate the Doublesex gene.…”