Alternative splicing of POUM2 regulates embryonic cuticular formation and tanning in Bombyx mori.

Insect cuticle is an apical extracellular matrix produced by the epidermis, tracheal, hind- and foregut epithelia during embryogenesis and renewed during moulting and metamorphosis. However, the underlined regulatory mechanism for embryonic cuticle formation remains largely unclear. Here, we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori, a model lepidopteran insect. CRISPR/Cas9-mediated knockout of POUM2 resulted in the defect of cuticular deposition, pigmentation and sclerotization in the embryos. Differentially expressed transcripts analysis of 7-day-old embryos identified 174 up- or down-regulated cuticular protein transcripts, eight up-regulated chitin degradation transcripts, two down-regulated chitin synthesis transcripts and 48 up- or down-regulated transcription factor transcripts in the POUM2 -/- embryos. The expression levels of the key factors of the tyrosine metabolic pathway, such as tyrosine hydroxylase (Th), Dopa decarboxylase (DDC) and arylalkylamine N-acetyltransferase (aaNAT), were significantly decreased in the POUM2 -/- embryos. POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element (CRE) in the Th promoter and increased the transcription of Th, whereas POUM2-S could not bound the POU CRE, although it also increased the transcription of Th. Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA. These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism, and the cuticular pigmentation and sclerotization by regulating the tyrosine metabolism during embryogenesis. This study provides new insights into novel function of POUM2 in embryogenesis. This article is protected by copyright. All rights reserved.