Matrin3 regulates mitotic spindle dynamics by controlling alternative splicing of CDC14B [Iso-Seq]

Purpose: Matrin3 (MATR3) is a DNA and RNA-binding protein and one of the major components of the nuclear matrix. Matrin3 is upregulated in colorectal cancer (CRC) compared to normal tissues, indicating potential oncogenic function in CRC. We found that depletion of Matrin3 results in decreased proliferation and colony formation in two CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates these effects, we aimed to find Matrin3 direct RNA targets and the effects of Matrin3 on them. Methods: We performed PAR-CLIP for endogenous Matrin3 and RNA-seq after Matrin3 silencing in HCT116 cells, a colorectal cancer cell line. Results: We uncovered Matrin3-mediated regulation of spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3 target RNAs transcriptome-wide in CRC cells, and found that Matrin3 broadly regulates alternative splicing. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss resulted in misprocessing of the CDC14B transcript that has a premature termination codon and simultaneous down-regulation of the standard CDC14B transcript in our model. Selective knockdown of the CDC14B standard variant phenocopied the loss of Matrin3 and resulted in reduced CRC cell proliferation, stabilized microtubules, and defects in mitotic spindle formation with tumbled mitotic spindles, suggesting that CDC14B is a key downstream effector of Matrin3. Conclusions: Our data show that by regulating the abundance of CDC14B standard variant, Matrin3 contributes to maintenance of microtubules dynamics, spindle morphology and proper mitotic spindle orientation and suggest that defects in this pathway may contribute to the reduced proliferation of cells after depletion of Matrin3. Iso-seq of HCT116 cell line

研究目的：Matrin3（MATR3）是一种DNA与RNA结合蛋白，同时也是核基质的核心组分之一。相较于正常组织，Matrin3在结直肠癌（colorectal cancer, CRC）中的表达水平显著上调，提示其在结直肠癌中可能发挥致癌功能。我们发现，敲低Matrin3会导致两株结直肠癌细胞系的增殖能力与集落形成能力显著下降。为阐明Matrin3介导上述效应的分子机制，我们旨在鉴定Matrin3的直接RNA靶标，并探究Matrin3对这些靶标的调控作用。
实验方法：我们在结直肠癌细胞系HCT116中，对内源性Matrin3开展了光活性增强的核糖核苷交联免疫沉淀（PAR-CLIP）实验，并在Matrin3沉默后进行了RNA测序（RNA-seq）。
实验结果：我们揭示了Matrin3对结直肠癌细胞纺锤体动态的调控作用。我们在结直肠癌细胞的全转录组范围内鉴定了被Matrin3结合并调控的靶RNA，同时发现Matrin3可广泛调控可变剪接事件。在排名靠前的Matrin3靶标中，我们聚焦于CDC14B，发现在本研究模型中，Matrin3缺失会导致携带提前终止密码子的CDC14B转录本加工异常，同时使标准型CDC14B转录本的表达水平下调。选择性敲低CDC14B标准亚型可模拟Matrin3缺失的表型，导致结直肠癌细胞增殖能力下降、微管稳定化，以及有丝分裂纺锤体紊乱、形态异常等纺锤体形成缺陷，这表明CDC14B是Matrin3的关键下游效应分子。
研究结论：我们的研究数据表明，Matrin3通过调控CDC14B标准亚型的表达丰度，维持微管动态平衡、纺锤体形态以及正常的有丝分裂纺锤体定向；该通路的功能缺陷可能是Matrin3缺失后细胞增殖能力下降的潜在原因之一。
HCT116细胞系的全长转录组测序（Iso-seq）