m6A 读取蛋白、IGF2BP2 和 HNRNPA2B1 对 N6-甲基腺苷修饰的 H19 lncRNA 处理的不同要求稳定性与 miR-675 的生物生成

bioRxiv Pub Date : 2024-08-08 DOI:10.1101/2024.08.07.606971
Samarjit Jana, Abhishek Chowdhury, K. Somasundaram
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引用次数: 0

摘要

H19是一种编码miR-675的lnc-pri-miRNA,在许多癌症中都存在失调。然而,H19加工,特别是miR-675形成的具体机制仍不清楚。我们的研究发现,在胶质母细胞瘤(GBM)和胶质瘤干细胞(GSCs)中,H19以一种依赖于METTL3的方式高表达并被m6A修饰。沉默METTL3会降低H19和miR-675的水平,而过表达METTL3会促进miR-675的处理,但不会影响H19的水平。此外,与 H19 水平相比,来自外源表达的 H19 的 miR-675 在沉默 METTL3 的胶质瘤细胞中受到的影响要大得多,这表明 m6A 修饰的 H19 转录本在处理过程中有不同的要求。我们证明,H19 与 m6A 阅读蛋白、IGF2BP2 和 HNRNPA2B1 相互作用,沉默这两种蛋白会降低 H19 和 miR-675 的水平。然而,与 IGF2BP2 沉默的胶质瘤细胞相比,HNRNPA2B1 沉默的胶质瘤细胞中 METTL3 过表达细胞中的高水平 miR-675 受到严重影响。有趣的是,IGF2BP2 沉默对外源 H19 构建物的 H19 稳定性影响更大,而 HNRNPA2B1 沉默则严重影响 miR-675 的处理。定点突变证实了在 H19 的第一个外显子中存在两个 m6A 位点,其中 1 号位点有利于 HNRNPA2B1 的相互作用,从而促进 miR-675 的加工。相反,2 号位点促进了 IGF2BP2 的相互作用,从而增强了 H19 的稳定性。H19-METTL3-HNRNPA2B1-miR675轴抑制miR-675的已知靶标Calneuron 1(CALN1),从而促进胶质瘤细胞迁移。值得注意的是,低CALN1/高H19预示着GBM患者的不良预后,而高METTL3或HNRNPA2B1而非IGF2BP2转录物水平会进一步加剧这种不良预后。因此,我们发现,H19转录本在GBM中高表达,并被m6A修饰,而m6A读取蛋白、IGF2BP2和HNRNPA2B1以不同方式调控H19的处理,以促进胶质瘤细胞迁移。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Differential requirement of m6A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N6-methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis
H19, a lnc-pri-miRNA that encodes miR-675, is dysregulated in numerous cancers. However, the specific mechanisms underlying H19 processing, particularly miR-675 formation, remain unclear. Our study reveals that H19 is highly expressed and m6A modified in a METTL3-dependent manner in glioblastoma (GBM) and glioma stem cells (GSCs). Silencing METTL3 reduced both H19 and miR-675 levels, whereas overexpressing METTL3 promoted miR-675 processing without affecting H19 levels. Further, miR-675 derived from exogenously expressed H19 was affected considerably more in METTL3 silenced glioma cells compared to H19 levels, suggesting differential requirements in the processing of m6A modified H19 transcript. We demonstrate that H19 interacts with m6A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either reduced H19 and miR-675 levels. However, a high level of miR-675 seen in METTL3 overexpressing cells is severely affected in HNRNPA2B1-silenced compared to IGF2BP2-silenced glioma cells. Interestingly, IGF2BP2 silencing more significantly affected H19 stability from exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Site-directed mutagenesis confirmed the presence of two m6A sites in the first exon of H19, with site #1 facilitating HNRNPA2B1 interaction to promote miR-675 processing. In contrast, the IGF2BP2 interaction is promoted by site #2, resulting in enhanced H19 stability. H19-METTL3-HNRNPA2B1-miR675 axis inhibited Calneuron 1 (CALN1), a known target of miR-675, to promote glioma cell migration. Notably, a low CALN1/high H19 predicted a poor prognosis in GBM patients and was further exacerbated by a high METTL3 or HNRNPA2B1 but not IGF2BP2 transcript levels. Thus, we found that the H19 transcript is highly expressed in GBM and m6A modified, and the m6A reader proteins, IGF2BP2 and HNRNPA2B1, regulate the H19 processing differently to promote glioma cell migration.
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