Abstract
Serine/arginine-rich proteins are a class of highly conserved splicing factor proteins involved in constitutive and alternative splicing. We screened a low molecular weight serine/arginine rich protein from silkworms and named it BmUP. Temporal and spatial expression analysis indicated that the BmUP gene was specifically expressed in the silkworm testis, and the highest expression occurred in the pre-pupa stage from the fifth instar to the moth stages. Here, we generated BmUP knockout individuals with the CRISPR/Cas9 system. Both the internal and external genitalia of knockout individuals were abnormal in knockout compared with wild-type male silkworms. In transgenic silkworms overexpressing BmUP, male silkworms showed a phenotype similar to that of the knockout individuals, whereas female individuals showed no significant differences from the wild type. In addition, by conducting promoter analysis, we identified Bmachi, a transcription factor that regulates the BmUP gene. Gel migration experiments revealed that BmAchi specifically binds the BmUP promoter. Quantitative real-time PCR showed that an increase in Bmachi expression up-regulated the expression of BmUP. In contrast, when the expression of Bmachi decreased, the expression of BmUP also downregulated in the experimental group compared with the control group. These results provide new insights for studying the effects of serine/arginine-rich proteins on the development of silkworm genitals.
Highlights
In most higher eukaryotes, splicing of precursor RNA is an essential step in the expression of protein coding genes
No prior research has described the relative expression of the BmUP gene in silkworms
To understand the temporal and spatial expression patterns of the BmUP gene in detail, we used semi-quantitative PCR and real-time fluorescence quantitative PCR to analyze the expression of BmUP in the gonads, silk glands, fat bodies, epidermis, Malpighian tubes and heads of female and male silkworms
Summary
In most higher eukaryotes, splicing of precursor RNA is an essential step in the expression of protein coding genes. The splicing process involves a large molecular complex that removes introns from the precursor RNA to form mature RNA. In this complex, the spliceosome is composed of nucleoprotein particles and many other proteins [4]. Serine/arginine-rich proteins, such as U2AF35, U2AF65 and U1-70K, have important roles [5]. SR proteins are a family of serine/arginine-rich proteins characterized by their RS structural domain. The discovery of these proteins arose from the identification of Drosophila splicing factors SWAP (suppressor of white-apricot) [7], Tra (transformer) [8] and Tra-2 (transformer-2) [9].
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