Alternative Polyadenylation Allows Differential Negative Feedback of Human miRNA miR-579 on Its Host Gene ZFR

About half of the known miRNA genes are located within protein-coding host genes, and are thus subject to co-transcription. Accumulating data indicate that this coupling may be an intrinsic mechanism to directly regulate the host gene’s expression, constituting a negative feedback loop. Inevitably, the cell requires a yet largely unknown repertoire of methods to regulate this control mechanism. We propose APA as one possible mechanism by which negative feedback of intronic miRNA on their host genes might be regulated. Using in-silico analyses, we found that host genes that contain seed matching sites for their intronic miRNAs yield longer 32UTRs with more polyadenylation sites. Additionally, the distribution of polyadenylation signals differed significantly between these host genes and host genes of miRNAs that do not contain potential miRNA binding sites. We then transferred these in-silico results to a biological example and investigated the relationship between ZFR and its intronic miRNA miR-579 in a U87 cell line model. We found that ZFR is targeted by its intronic miRNA miR-579 and that alternative polyadenylation allows differential targeting. We additionally used bioinformatics analyses and RNA-Seq to evaluate a potential cross-talk between intronic miRNAs and alternative polyadenylation. CPSF2, a gene previously associated with alternative polyadenylation signal recognition, might be linked to intronic miRNA negative feedback by altering polyadenylation signal utilization.

已知的微小RNA（miRNA）基因中约有半数位于编码蛋白的宿主基因内，因而与宿主基因发生共转录。越来越多的研究数据显示，这种共转录耦合或许是直接调控宿主基因表达的内在机制，进而形成负反馈环路。毋庸置疑，细胞需要一套迄今仍知之甚少的调控手段集合，以管控这一调控机制。我们提出可变聚腺苷酸化（Alternative Polyadenylation, APA）或许是调控内含子miRNA对宿主基因产生负反馈的潜在机制之一。通过计算机模拟（in silico）分析，我们发现携带自身内含子miRNA种子序列匹配位点的宿主基因，其3'非翻译区（3'UTR）更长，且包含更多聚腺苷酸化位点。此外，相较于不携带潜在miRNA结合位点的宿主基因，这类宿主基因的聚腺苷酸化信号分布存在显著差异。随后，我们将上述计算机模拟的研究结果拓展至生物学实例，在U87细胞系模型中探究了ZFR与其内含子miRNA miR-579之间的调控关系。研究证实，ZFR会被其内含子miRNA miR-579靶向，且可变聚腺苷酸化可介导差异化的靶向调控。此外，我们还借助生物信息学分析与RNA测序（RNA-Seq），评估了内含子miRNA与可变聚腺苷酸化之间潜在的分子串扰。此前被证实与可变聚腺苷酸化信号识别相关的切割与多聚腺苷酸化特异性因子2（CPSF2），或可通过改变聚腺苷酸化信号的利用效率，参与内含子miRNA介导的负反馈调控过程。