Identification of a LncRNA EPR—>METTL7A1 pathway modulating translation of select genes (Total RNA)

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription and, here, we report on Mettl7a1 as a target of EPR. We show that the lncRNA induces Mettl7a1 transcription by remodeling the 3-dimensional chromatin structure at the Mettl7a1 locus. Our data indicate that METTL7A1 participates in the EPR-dependent pathway that antagonizes TGF-ß signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METT7A) is downregulated in breast cancers. Importantly, expression of METTL7A1 in 4T1 tumorigenic cells reduces their transformation potential, and the putative methyltransferase activity of METTL7A1 appears dispensable for its biological functions. METTL7A1 is a cytoplasmic protein and, from a mechanistic perspective, interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of select proteins without substantial changes in mRNA abundance. Our data suggest the possibility that METTL7A1 conveys the transcriptional regulation operated by EPR into specific changes of mRNA translation. Overall design: High-quality RNA was extracted from either mock or METTL7A1-overexpressing 4T1 cells (biological triplicates for each experimental condition), and a total of six libraries were prepared using standard Illumina Stranded Total RNA Prep with Ribo-Zero Plus protocol and sequenced on Illumina NovaSeq 6000.

EPR是一种长链非编码RNA（long non-coding RNA，lncRNA），可通过调控基因转录控制乳腺细胞的增殖。本研究报道Mettl7a1为EPR的靶基因：该lncRNA通过重塑Mettl7a1基因座的三维染色质结构，诱导Mettl7a1的转录。实验数据表明，METTL7A1参与EPR依赖的、拮抗转化生长因子β（transforming growth factor β，TGF-β）信号通路的过程。致瘤性小鼠乳腺细胞中不存在METTL7A1，其人类同源基因METT7A在乳腺癌中表达下调。值得关注的是，在4T1致瘤细胞中过表达METTL7A1可降低其转化潜能，且METTL7A1的推定甲基转移酶活性对其生物学功能似乎并非必需。METTL7A1是一种细胞质蛋白；从机制角度而言，它可与mRNA翻译早期阶段相关的因子相互作用，结合核糖体，并在不显著改变mRNA丰度的情况下，影响特定蛋白质的表达水平。本研究数据提示，METTL7A1可能将EPR介导的转录调控转化为mRNA翻译的特异性改变。

实验整体设计：从空白对照（mock）组与过表达METTL7A1的4T1细胞（每个实验条件设置三组生物学重复）中提取高质量RNA，采用标准Illumina Stranded Total RNA Prep with Ribo-Zero Plus建库流程构建文库，总计构建6个文库，并在Illumina NovaSeq 6000测序平台上完成测序。