ADAR1於干擾素訊號路徑中角色的研究與探討

Abstract

ADAR1為一具有脫氨活性的酵素，能將雙股RNA上與胸苷酸配對的腺苷酸進行脫氨而形成肌苷酸。由於肌苷酸結構與鳥苷酸相似，這種核苷酸序列的改變可能影響到RNA的二級結構、RNA的穩定性甚至是密碼子的組成，造成基因表現程度甚至是基因功能的改變。ADAR1包含兩種蛋白異構體，分別為ADAR1p150和ADAR1p110，其中ADAR1p150的表現會受到干擾素的訊號路徑刺激而增加，此外許多文獻指出病毒的RNA也會受到ADAR1的修飾。因此ADAR1被認為參與在干擾素的相關路徑當中，但是目前對於ADAR1在其中扮演的角色仍舊不是很清楚。在本篇研究中，我們利用siRNA抑制ADAR1的表現，證實IFI27L1、 IFNAR1、 IFNAR2、 MAVS、 RIG-1皆受到ADAR1的修飾，其中IFI27L1的mRNA有增加的趨勢。另一方面，PRKRA的mRNA和蛋白質表現量有明顯的下降，而根據次世代定序資料庫的資訊，我們證實在PRKRA轉錄本的下游有一段與之重疊的反義轉錄本，同時此反義轉錄本也會受到ADAR1的修飾。我們發現當ADAR1的表現被抑制後，此反義轉錄本的表現量以及RNA半生期都有增加的趨勢。綜合上述結果，我們發現許多參與在RLR以及JAK/STAT訊號路徑的基因，都會受到ADAR1的修飾；此外我們推測ADAR1能藉由與反義轉錄本的相互作用，進而影響到相關基因的表現。

ADAR1 enzyme catalyzes deamination of adenine(A) on double stranded RNA to yield inosine(I). Because inosine is recognized as guanine by cellular machineries, such ADAR1-meidated post-transcriptional nucleotide sequence modification can affect several gene expressions and biological processes. Several studies have demonstrated that, in addition to cellular targets, viral transcripts are substrates for ADAR1, and that ADAR1 exhibits both anti-viral and proviral activities. Interferon (IFN), acting as an antiviral agent, is known to induce the expression of ADAR1 p150 isoform, further suggesting that ADAR1 may be linked to the IFN signaling pathway; however, its roles in this regulation remains uncharacterized. Our recent deep sequencing approach of identifying gene targets of ADAR1 revealed several candidate genes with functions associated with RLR and JAK/STAT signaling. We tested and confirmed editing events and RNA expression level of IFI27L1, IFNAR1, IFNAR2, MAVS, RIG-1 and PRKRA. The A-to-I editing events are confirmed by Sanger sequencing, which further showed obvious ADAR1-dependent editing events in IFI27L1, IFNAR1, IFNAR2, MAVS and RIG-1. Furthermore, higher RNA expression level of IFI27L1 is detected in ADAR1 knockdown cells. With regard to the PRKRA transcript, our observation and sequencing data further imply that there is an anti-sense transcript overlapping with 3’ end of the PRKRA gene locus and that RNA editing may be linked to its regulation. Intriguingly, both mRNA as well as protein expression are decreased and half-life time of the antisense transcript is increased after ADAR1 knockdown. Based on these preliminary findings, we hypothesize that ADAR1 may affect these genes at post-transcriptional level, and in turn modulate the associated signaling pathway. Future work focused on dissecting the mechanism of ADAR1 function in RLR and JAK/STAT signaling as well as in the regulation of PRKRA expression is discussed.