Characterization of nuclear localization signal in Ostrinia furnacalis Masculinizer protein

Abstract

Bombyx mori Masculinizer protein (BmMasc) is essential for both masculinization and dosage compensation in B. mori. We previously identified a bipartite nuclear localization signal (NLS) of BmMasc and two essential residues (lysine at 274 [K274] and arginine at 275 [R275]) implicated in its function. Sequence comparison showed the presence of putative NLSs in lepidopteran Masc proteins, but their functional properties and critical residues are unknown. Here we characterized a putative NLS of Ostrinia furnacalis Masc (OfMasc) using B. mori ovary‐derived BmN‐4 cell line. Deletion and alanine scanning mutagenesis revealed that a putative NLS is required for nuclear localization of OfMasc. However, mutations at both K227 and R228, which correspond to K274 and R275 of BmMasc, respectively, do not greatly abolish the NLS activity. Additional mutagenesis analysis revealed that triple mutations at K227, R228, and K240 almost completely inhibited OfMasc nuclear localization. These results suggest that lepidopteran Masc proteins possess a common functional NLS, but the critical residues for its activity are different. Moreover, we examined the masculinizing activity of OfMasc derivatives and found that nuclear localization is not required for the masculinizing activity of OfMasc. The results from our studies indicate that lepidopteran Masc proteins function in the cytoplasm to drive masculinizing cascade.

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“…The codon-optimized Oscar , wmk1 , wmk2 , and wmk3 fragments were synthesized by Eurofins Genomics (Germany). The DNA fragments of OfMasc-GFP 26 , wmk1 , wmk2 , wmk3 , Oscar , and their derivatives were cloned into the pIZ/V5-His-g3 vector 26 . Trilocha varians Masc ( TvMasc ) 27 , S. frugiperda Masc ( SfMasc ), and Papilio machaon Masc ( PmMasc ) cDNAs fused to EGFP fragment were also cloned into the pIZ/V5-His-g3 vector.…”