A novel antisense RNA ASPACT confers multi-level suppression of PACT and associated signalling

The innate immune system is the frontline host protection against pathogens. Effective antiviral immunity is elicited upon recognition of viral RNAs by the host pattern recognition receptors. One of the major viral RNA sensors is retinoic acid inducible gene-1, which triggers the production of interferons (IFNs). In turn, this protective response requires another viral sensor and immunity factor interferon-inducible protein kinase RNA activator (PACT/PRKRA). Here, we report the identification and characterization of a novel antisense <i>PACT</i> gene that expresses a non-coding RNA in a convergent and interferon-inducible manner. Publicly available gene structure and expression data revealed that this gene, that we termed <i>ASPACT</i>, overlaps with the 3′ -end of the <i>PACT</i> locus and is highly expressed during viral infection. Our results confirm the IFN-β-inducibility of <i>ASPACT</i>, which is dependent on STAT-1/2. We further discovered that downregulation of ASPACT impacts both the expression and localization of the PACT transcript. At the transcription level, ChIP and ChIRP assays demonstrated that the ASPACT non-coding RNA occupies distinct chromatin regions of <i>PACT</i> gene and is important for promoter recruitment of the epigenetic silencer HDAC1. In parallel, ASPACT was also found to mediate nuclear retention of the PACT mRNA via direct RNA–RNA interaction, as revealed by RNA antisense purification assay. In summary, our results support the model that the non-coding RNA ASPACT acts as a negative regulator of PACT at multiple levels, and reveal a novel regulator of the viral counteractive response.

先天免疫系统是宿主对抗病原体的第一道防线。宿主模式识别受体（pattern recognition receptors）识别病毒RNA后，可触发有效的抗病毒免疫应答。视黄酸诱导基因1（retinoic acid inducible gene-1）是主要的病毒RNA传感器之一，其可诱导干扰素（IFNs）的产生。而这一保护性应答的实现，还依赖于另一种病毒传感器及免疫因子——干扰素诱导蛋白激酶RNA激活因子（PACT/PRKRA）。本研究鉴定并表征了一种新型反义<i>PACT</i>基因，该基因以反向转录且干扰素诱导的方式表达一种非编码RNA（non-coding RNA）。公开可得的基因结构与表达数据显示，我们将该基因命名为<i>ASPACT</i>，其与<i>PACT</i>基因座的3'端区域存在重叠，且在病毒感染过程中呈高表达水平。我们的实验结果证实了<i>ASPACT</i>的β干扰素（IFN-β）诱导性，且该过程依赖于信号转导与转录激活因子1/2（STAT-1/2）。进一步研究发现，<i>ASPACT</i>的下调会同时影响<i>PACT</i>转录本的表达与定位。在转录水平上，染色质免疫沉淀（ChIP）与染色质寡核苷酸亲和纯化（ChIRP）实验证实，<i>ASPACT</i>非编码RNA可结合<i>PACT</i>基因的特定染色质区域，且对表观遗传沉默因子HDAC1在启动子区域的募集至关重要。同时，RNA反义纯化实验显示，<i>ASPACT</i>还可通过直接的RNA-RNA相互作用介导<i>PACT</i> mRNA的核滞留。综上，本研究结果支持<i>ASPACT</i>非编码RNA作为多层面的<i>PACT</i>负调控因子的模型，并揭示了病毒拮抗宿主免疫应答的一种新型调控因子。