Functional and evolutionary significance of human microRNA seed region mutations [M5]. Homo sapiens

MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in the regulation of gene expression at the post transcriptional and/or translational level thus impacting various biological processes. Dysregulation of miRNAs could affect processes associated with progression of a variety of diseases including cancer. Majority of miRNA targeting in animals involves a 7-nt “seed region” mapping to positions 2-8 at the molecule’s 5' end. The importance of this 7 nt sequence to miRNA function is evidenced by the fact that the seed region sequence of many miRNAs is highly conserved within and between species. In this study, we computationally and experimentally explore the functional significance of sequence variation within the seed region of human miRNAs. Our results indicate that change of a single nt within the 7-nt seed region changes the spectrum of targeted mRNAs significantly meanwhile further nt changes have little to no additional effect. This high functional cost of even a single nucleotide change within the seed region of miRNAs explains why the seed sequence is highly conserved among many miRNA families both within and between species and could help clarify the likely mechanisms underlying the evolution of miRNA regulatory control. Overall design: mRNAs were collected from 3 M5 miRNA treated and 3 negative control miRNA treated HEY ovarian cancer cell samples. mRNA expression was captured on Affymetrix U133 Plus 2 chips. To compare mRNA expression pattern between the M5 treated cells and the negative control treated cells, present/absent calls were generated using MAS5, while signals were calculated using GCRMA and then log2 transformed. Expression of differentially expressed genes or down regulated miRanda-mirSVR predicted miRNA target genes was compared between miRNA treated samples.

微小RNA（microRNAs, miRNAs）是一类小型非编码RNA，在转录后及/或翻译水平的基因表达调控中发挥核心作用，进而影响诸多生物学过程。miRNAs的表达失调可影响包括癌症在内的多种疾病的进展相关过程。在动物体内，大多数miRNA的靶向调控过程依赖一段位于其分子5'端2-8位的7核苷酸“种子区域（seed region）”。诸多miRNA的种子区域序列在物种内及跨物种间均高度保守，这一事实充分佐证了该7核苷酸序列对miRNA功能的重要性。本研究通过计算与实验手段，探究了人类miRNA种子区域内序列变异的功能意义。本研究结果显示，7核苷酸种子区域内单个核苷酸的改变即可显著改变其靶向mRNA的靶标谱，而后续额外的核苷酸改变几乎不会带来额外影响。即便miRNA种子区域内单个核苷酸的改变也会带来如此高的功能代价，这一现象解释了为何诸多miRNA家族的种子序列在物种内及跨物种间均高度保守，同时也有助于阐明miRNA调控体系演化背后的潜在机制。实验整体设计：从3份经M5 miRNA处理及3份经阴性对照miRNA处理的HEY卵巢癌细胞样本中提取信使RNA（messenger RNA, mRNA）；使用Affymetrix U133 Plus 2芯片完成mRNA表达谱的检测。为比较M5处理组与阴性对照组的mRNA表达模式，研究人员使用MAS5算法生成存在/缺失检出判定结果，同时采用GCRMA算法计算信号强度并进行log2对数转换。在经miRNA处理的各组样本间，比较差异表达基因或经miRanda-mirSVR预测得到的下调miRNA靶基因的表达水平。