Alternative Splicing of bunched Confers a Dual Role in Hippo Pathway-dependent Growth and Tumorigenesis

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. BunA with HA tag was ectopically expressed under the promotor of nub-Gal4, and the anti-HA antibody was used to enrich the DNA fragments bound by BunA

可变剪接（Alternative splicing）是一类核心分子机制，可从单个基因编码产生功能各异的蛋白质，对基因调控与蛋白质组多样性的形成具有关键贡献。在果蝇（Drosophila）中，bunched（bun）基因作为TSC-22结构域基因家族（TSC-22 domain gene family）的成员，可发生可变剪接，生成参与诸多关键生物学过程的多种蛋白质异构体。然而，各蛋白质异构体的具体功能及调控机制仍未明确。本研究借助CRISPR/Cas9技术，在bun基因的内源基因座上引入靶向缺失突变，分别实现长、短型蛋白质异构体的定向移除。实验发现，短型异构体表现出生长抑制作用，而长型异构体则具备促生长效应。令人意外的是，长型异构体展现出显著的双重功能：无论是敲除还是过表达长型异构体，均会阻碍Hippo通路（Hippo pathway）失活所诱导的过度生长。从机制层面而言，异位表达的Bun长型异构体可作为转录抑制因子，通过与Yorkie/Scalloped（Yki/Sd）竞争性结合靶基因的启动子区域，抑制其转录输出，最终介导生长抑制。本研究揭示了Bun不同剪接异构体与致癌表型之间的复杂相互作用，明确了Bun长型异构体作为关键转录抑制因子，在果蝇体内调控Hippo通路失活介导的生长异常与肿瘤发生过程。此外，本研究在nub-Gal4启动子驱动下异位表达了带有HA标签（HA tag）的BunA，并使用抗HA抗体富集被BunA结合的DNA片段。