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Updated: May 28, 2026

Using the E1A Minigene Tool to Study mRNA Splicing Changes
Published on: April 22, 2021
Effects of APRIL (TNFSF13) polymorphisms and splicing isoforms on the secretion of soluble APRIL
Takumi Furuya1, Minori Koga, Koki Hikami
1Molecular and Genetic Epidemiology Laboratory, Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.
Abstract:
Functional APRIL (TNFSF13) is a secreted trimer generated by furin protease cleavage. We previously reported the association of APRIL haplotypes formed by two nonsynonymous polymorphisms, Gly67Arg and Asn96Ser, with systemic lupus erythematosus. Here, we tested the hypothesis that polymorphisms and/or alternative splicing may influence the generation of soluble APRIL (sAPRIL). HEK 293T cells were transfected with plasmids containing one of the six combinations of splicing isoforms (α or β) and haplotypes (susceptible, neutral, or protective). APRIL concentrations were quantitated in the cell lysates and supernatants using an enzyme-linked immunosorbent assay (ELISA). The association between splicing efficiency and polymorphisms was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR). The efficiency of cleavage by furin protease was analyzed using western blotting. Although both splicing isoforms were cleaved by furin protease, sAPRIL was not detected in the supernatant of the cells transfected with the β isoform, regardless of the haplotype. This suggested that, similarly to B-cell activating factor (BAFF), one of the major APRIL splicing isoforms may not be secreted as a functional molecule. Furthermore, the secretion of sAPRIL was decreased in the transfectants expressing the protective haplotype. An association between the polymorphisms and splicing efficiency or furin cleavage efficiency was not detected. In conclusion, these observations suggested that both alternative splicing and polymorphisms may affect the generation of functional sAPRIL.
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