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Alternative splicing of bunched confers a dual role in hippo pathway-dependent growth and tumorigenesis

Oncogene volume 44, pages 1949–1960 (2025)Cite this article

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Abstract

Alternative splicing is a fundamental mechanism that generates functionally distinct proteins from individual genes, contributing to gene regulation and proteomic diversity. In Drosophila, the bunched (bun) gene, a member of the TSC-22 domain gene family, undergoes alternative splicing, yielding diverse protein isoforms involved in crucial biological processes. Nevertheless, the specific roles and regulatory mechanisms of each isoform remain elusive. Here, we employed CRISPR/Cas9 technology to introduce targeted deletions within the endogenous locus of the bun gene, resulting in the removal of either long or short isoforms. We discovered that the short isoforms demonstrated a growth-suppressive role, whereas the long isoforms exhibited a growth-promoting effect. Surprisingly, the long isoforms exhibited a remarkable dual functionality, as both deletion and amplification of long isoform expression impede the excess growth induced by Hippo pathway inactivation. Mechanistically, ectopically expressed Bun long isoforms act as the transcriptional suppressor by competitively binding to targets’ promoter regions in conjunction with Yorkie/Scalloped (Yki/Sd), thereby inhibiting its transcriptional outputs and ultimately leading to the growth suppression. These findings unveil the intricate interaction between distinct spliced isoforms of Bun and oncogenic outcomes, highlighting Bun long isoforms as the critical transcription suppressor regulating Hippo pathway inactivation-mediated growth and tumorigenesis in Drosophila.

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Fig. 1: Phenotypic characterization of splicing specific bun mutants.
Fig. 2: Ectopic expression of long isoform BunA exhibits dual roles in different genetic backgrounds.
Fig. 3: The long isoform BunA competes with Sd for binding to the Hippo pathway targets.
Fig. 4: BunA overexpression restricts tissue growth and represses the transcription activation of Hippo pathway target genes.
Fig. 5: BunA overexpression inhibits Hippo signaling inactivation-induced overgrowth and tumorigenesis.

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Data availability

Raw sequencing data for the three BunA CUT&Tag replicates has been submitted to the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274246) under the accession number of GSE274246.

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Acknowledgements

We thank Tian Xu, Lei Xue, Bloomington Drosophila Stock Center, Vienna Drosophila Resource Center, TsingHua Fly Center, and DSHB for providing fly stocks and reagents. We thank Wenhan Liu from our lab for fly stock maintenance. Cartoon illustrations in the manuscript were created with BioRender.com. This work was supported by startup funds from Westlake University9uj and grants from the National Natural Science Foundation of China (32170824, 32322027) to XM and (32370710) to YZ, HRHI program (1011103360222B1) of Westlake Laboratory of Life Sciences and Biomedicine to XM, Westlake Laboratory of Life Sciences and Biomedicine (10128A092001), and “Team for Growth Control and Size Innovative Research” (201804016). YZ is also supported by the National Key Research and Development Program (2022YFA1302700).

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Author notes

  1. These authors contributed equally: Pengjuan Guo, Sha Song, Yuxiao Niu.

Authors and Affiliations

  1. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, 310024, China

    Pengjuan Guo, Sha Song, Yuxiao Niu, Xiaoyu Kuang, Yanxiao Zhang & Xianjue Ma

  2. School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China

    Pengjuan Guo, Sha Song, Yuxiao Niu, Xiaoyu Kuang, Yanxiao Zhang & Xianjue Ma

  3. College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong, 271018, China

    Dafa Zhou & Zizhang Zhou

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Contributions

XM conceived and designed the study; XM, YZ, and PG designed the experiments and analyzed the data; PG performed the majority of the experiments with the help and input from SS, DZ, XK, and ZZ; YN performed CUT&Tag analyses; XM, SS, XK, and PG wrote the original manuscript; XM, PG, and SS wrote the revised manuscript.

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Correspondence to Yanxiao Zhang or Xianjue Ma.

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Guo, P., Song, S., Niu, Y. et al. Alternative splicing of bunched confers a dual role in hippo pathway-dependent growth and tumorigenesis. Oncogene 44, 1949–1960 (2025). https://doi.org/10.1038/s41388-025-03348-6

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