Sahu Shriya, Williams Leo, Perez Alberto, Philip Finly, Caso Giuseppe, Zurawsky Walter, Scarlata Suzanne
Department of Physiology & Biophysics, Stony Brook University, Stony Brook, New York.
Laufer Center for Computational Biology, Stony Brook University, Stony Brook, New York.
Protein Sci. 2017 Sep;26(9):1807-1818. doi: 10.1002/pro.3219. Epub 2017 Jul 17.
RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cβ, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 10 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCβ. This finding is supported by microarray analysis in cells over-expressing PLCβ1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cβ/calcium signaling pathway.
RNA诱导沉默是一个使细胞能够调节特定蛋白质合成的过程。RNA沉默由蛋白质C3PO（RNA诱导沉默复合体的组分3）促进。我们之前发现，磷脂酶Cβ可响应特定G蛋白信号增加细胞内钙水平，它会抑制C3PO对某些基因的活性。了解控制C3PO活性的参数以及哪些基因受G蛋白激活影响，将有助于预测哪些基因更容易受到下调影响。在此，我们使用一个包含10种寡核苷酸的文库，表明C3PO以结构特异性而非序列特异性结合寡核苷酸。另外，C3PO水解寡核苷酸的速率对底物稳定性敏感。重要的是，我们发现具有较高解链温度（Tm）值的寡核苷酸会被结合的PLCβ抑制。在过表达PLCβ1的细胞中进行的微阵列分析支持了这一发现。综上所述，这项研究能够预测那些转录后调控对G蛋白/磷脂酶Cβ/钙信号通路有响应的基因。
