Long Non-Coding RNA Bmdsx-AS1 Effects on Male External Genital Development in Silkworm

SIMPLE SUMMARY: LncRNAs are a class of non-coding RNAs longer than 200 nt that are involved in a variety of biological processes. Studies on lncRNAs in Bombyx mori have shown that some lncRNAs are involved in brain development, silk production and the response to virus infection of the host. However, the roles of lncRNAs are still largely unknown in the silkworm. In this study, we analyzed the function of lncRNAs Bmdsx-AS1 in silkworm by transgenic overexpression, which not only affects the development of male silkworm external genitalia, but also participates in the regulation of EGFR signaling pathway. Moreover, we studied the upstream promoter of Bmdsx-AS1 and found that the BmAbd-B transcription factor of the Hox gene family can negatively regulate the expression of Bmdsx-AS1. These results laid a substantial foundation for in-depth study of the function of lncRNAs in the silkworm. ABSTRACT: Long non-coding RNAs (lncRNAs) have been suggested to play important roles in some biological processes. However, the detailed mechanisms are not fully understood. We previously identified an antisense lncRNA, Bmdsx-AS1, that is involved in pre-mRNA splicing of the sex-determining gene Bmdsx in the silkworm. In this study, we analyzed the changes in the male external genitalia of transgenic overexpressed Bmdsx-AS1 silkworm lines and analyzed downstream and upstream responses. We found that Bmdsx-AS1 transgenic silkworms, compared with wild type, showed more claspers in the male external genitalia. Quantitative real-time PCR (qPCR) results indicated that overexpression of Bmdsx-AS1 decreased the expression of genes in the EGFR signaling pathway. Knockdown of Bmdsx-AS1 increased the activity of the EGFR pathway. Through promoter prediction, promoter truncation and electrophoretic mobility shift assay (EMSA) analyses, we found that the protein encoded by the Hox gene BmAbd-B specifically binds to the promoter of Bmdsx-AS1. Moreover, overexpression of BmAbd-B in the silkworm BmE cell line indicated that BmAbd-B negatively regulates the mRNA expression of Bmdsx-AS1. Our study provides insights into the regulatory mechanism of the lncRNA in the silkworm.