Ghosal Suman, Saha Shekhar, Das Shaoli, Sen Rituparno, Goswami Swagata, Jana Siddhartha S, Chakrabarti Jayprokas
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata, West Bengal, 700032, India.
Gyanxet, BF 286 Salt Lake, Kolkata, West Bengal, 700064, India.
Sci Rep. 2016 Feb 29;6:22334. doi: 10.1038/srep22334.
Some earlier studies have reported an alternative mode of microRNA-target interaction. We detected target regions within mRNA transcripts from AGO PAR-CLIP that did not contain any conventional microRNA seed pairing but only had non-conventional binding sites with microRNA 3' end. Our study from 7 set of data that measured global protein fold change after microRNA transfection pointed towards the association of target protein fold change with 6-mer and 7-mer target sites involving microRNA 3' end. We developed a model to predict the degree of microRNA target regulation in terms of protein fold changes from the number of different conventional and non-conventional target sites present in the target, and found significant correlation of its output with protein expression changes. We validated the effect of non-conventional interactions with target by modulating the abundance of microRNA in a human breast cancer cell line MCF-7. The validation was done using luciferase assay and immunoblot analysis for our predicted non-conventional microRNA-target pair WNT1 (3' UTR) and miR-367-5p and immunoblot analysis for another predicted non-conventional microRNA-target pair MYH10 (coding region) and miR-181a-5p. Both experiments showed inhibition of targets by transfection of microRNA mimics that were predicted to have only non-conventional sites.
一些早期研究报道了一种替代性的微小RNA-靶标相互作用模式。我们在AGO PAR-CLIP的mRNA转录本中检测到了靶标区域，这些区域不包含任何传统的微小RNA种子配对，仅具有与微小RNA 3'端的非传统结合位点。我们对7组在微小RNA转染后测量全局蛋白质折叠变化的数据进行的研究表明，靶标蛋白质折叠变化与涉及微小RNA 3'端的6聚体和7聚体靶标位点相关。我们开发了一个模型，根据靶标中存在的不同传统和非传统靶标位点的数量来预测微小RNA靶标调控在蛋白质折叠变化方面的程度，并发现其输出与蛋白质表达变化具有显著相关性。我们通过调节人乳腺癌细胞系MCF-7中微小RNA的丰度，验证了与靶标的非传统相互作用的效果。使用荧光素酶测定法和免疫印迹分析对我们预测的非传统微小RNA-靶标对WNT1（3'UTR）和miR-367-5p进行验证，并使用免疫印迹分析对另一个预测的非传统微小RNA-靶标对MYH10（编码区）和miR-181a-5p进行验证。两个实验均显示，转染预计仅具有非传统位点的微小RNA模拟物可抑制靶标。
