Post-transcriptional Gene Silencing Mediated by microRNAs is Controlled by Nucleoplasmic Sfpq [RNA-Seq]. Mus musculus

There is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here, we show by quantitative proteomic analysis that Ago2 interacts with nucleoplasmic Sfpq in a RNA-dependent fashion. By HITS-CLIP and transcriptomic analyses, we demonstrated that Sfpq directly controls the miRNA targeting of a subset of crucial miRNA-target mRNAs when it binds locally. Sfpq modulates miRNA targeting in both nucleoplasm and cytoplasm, indicating a nucleoplasmic imprinting of Sfpq-target mRNAs that influence miRNA targeting in both cellular compartments. Mechanistically, Sfpq binds to a sizeable set of long 3’UTR forming long aggregates to optimize miRNA position/recruitment to selected binding sites, as we show for Lin28A mRNA. These results extend the miRNA-mediated post-transcriptional gene silencing into the nucleoplasm and indicate that an unique Sfpq-dependent post-transcriptional strategy for controlling both nuclear and cytoplasmic gene expression takes place in cells during physio-pathological events. Overall design: RNA-seq of P19 cells control and upon SFPQ knockdown both in triplicates

越来越多的证据表明，微小RNA诱导沉默复合体（miRISC）存在于哺乳动物细胞核内并具有生物学活性。本研究通过定量蛋白质组学分析证实，Argonaute 2（Ago2）以RNA依赖的方式与核质蛋白Sfpq发生相互作用。借助HITS-CLIP（交联免疫沉淀联合高通量测序）与转录组分析，我们证明当Sfpq发生局部结合时，可直接调控一类关键miRNA靶标信使RNA（mRNA）的miRNA靶向过程。Sfpq可在核质与细胞质中均调控miRNA靶向，这提示Sfpq靶标mRNA存在核质印记，可影响两个细胞区域内的miRNA靶向功能。机制层面，Sfpq可结合大量含长3'非翻译区（3’UTR）的转录本并形成大型聚集复合体，以优化miRNA对特定结合位点的定位与招募，这一点在Lin28A mRNA中已得到验证。本研究将miRNA介导的转录后基因沉默调控范围拓展至核质区域，并表明细胞在生理病理过程中存在一种独特的、依赖Sfpq的转录后调控策略，可同时调控细胞核与细胞质内的基因表达。实验整体设计：对SFPQ敲低及对照的P19细胞进行RNA测序，每组设置3次生物学重复。