LINE1 contributes to autoimmunity through both RIG-I- and MDA5-mediated RNA sensing pathways
Introduction
Autoimmunity represents an abnormal activation of the endogenous immune system and the production of interferon (IFN) in the absence of exogenous stimulation, such as viral infection. The study of autoimmune diseases has provided considerable information regarding the host's mechanisms to counteract autoimmunity; one of the best examples of such diseases is Aicardi-Goutières syndrome (AGS). Several factors, such as TREX1, RNaseH2, SAMHD1, and ADAR1, have been associated with AGS; mutations of these factors have been associated with elevated type I IFN levels in the cerebrospinal fluid of AGS patients [reviewed in Ref. [1]]. Interestingly, although the exact cause has not been identified for the disease, these AGS-associated factors share retroelements as common targets [[2], [3], [4], [5]].
Retroelements occupy approximately 40% of the human genome and belong to transposons that can change their positions within the genome. One of them, long interspersed element type 1 (LINE1 or L1), is the only known autonomous non-LTR retroelement that is responsible for the replication of not only LINE1 itself but also of other non-autonomous retroelements such as Alu and SVA [6,7]. LINE1-mediated retrotransposition requires a procedure termed target-site-primed reverse transcription (TPRT), which involves nicking of the genome and subsequent synthesis of retroelement cDNA [8,9]. Interestingly, both products of TPRT (i.e., LINE1 cDNA and nicked genome) are potential triggers of cGAS, an endogenous DNA sensor that activates STING as well as IFN production [[10], [11], [12], [13]]. Meanwhile, various studies have indicated that, in murine cells expressing AGS-associated TREX1 or RNaseH2 mutants, LINE1 cDNA levels are elevated, and the cGAS-STING pathway is activated [2,14], linking LINE1 activity to autoimmunity.
In theory, TREX1, SAMHD1, and RNaseH2 could suppress autoimmunity by compromising TPRT and reducing LINE1 cDNA synthesis with their known functions (DNA exonuclease, dNTP hydrolase, and ribonuclease H activities, respectively), and so could ADAR1 by introducing mutations to LINE1 proteins through RNA mutagenesis on LINE1 RNA [as reviewed in Ref. [4]]. Surprisingly however, some of TREX1, RNaseH2, or ADAR1 mutants detected on human AGS patients remain active for their enzyme activities [[15], [16], [17]], suggesting LINE1 TPRT may not be the only reason for LINE1 triggering innate immune activation. On the other hand, some of these AGS-associated factors inhibit LINE1 retrotransposition through distinct mechanisms. For instance, TREX1 and SAMHD1 suppress LINE1 activity by lowering levels of LINE1 ORF1p and ORF2p, respectively [3,17]. In addition, ADAR1 represses LINE1 through LINE1 RNA interaction but not its RNA mutagenesis activity [18]. LINE1 RNA, ORF1p, and ORF2p are essential components for the assembly of LINE1 ribonucleoprotein particle (RNP), and targeting LINE1 RNP appears to be a common feature of these AGS-associated proteins that regulate innate immune activation. It is therefore possible that, LINE1 may activate IFN production through additional mechanism(s), where the integrity of LINE1 RNP may be essential.
Section snippets
Plasmid construction
The ADAR1 gene (expressing ADAR1 isoform p110) was retrieved from HEK293T cells through reverse transcription followed by polymerase chain reaction and then subcloned into VR1012 [19,20] (containing a CMV promoter/enhancer, intronA, multiple cloning sites, and BGH polyA signal sequence) with an HA tag at the C-terminus. ADAR1 isoform p150 was excluded from all tests with exogenous ADAR1 expression because its expression requires IFN induction, whereas the present study was focused on the impact
Endogenous LINE1 activates IFNβ production
To first test whether endogenous LINE1 could affect innate immune activation, we introduced LINE1-specific siRNA targeting ORF1 or ORF2 regions of the LINE1 genome into THP-1 cells (Fig. 1A, and Fig. S1). Endogenous RNA of siRNA-treated THP-1 cells were extracted at 48 h post-transfection and quantified through quantitative real-time polymerase chain reaction (qRT-PCR). As a result, LINE1-specific siRNA potently reduced endogenous levels of LINE1 RNA in THP-1 cells (Fig. 1B). Consistent with
Discussion
In human cells, LINE1 is the only known retroelement that is competent for autonomous replication [46]. Retrotransposition, especially TPRT, involves genome nicking and cDNA synthesis [8,47]. Therefore, it has been hypothesized accordingly that LINE1 might activate IFN production through DNA sensing pathways [4]. Surprisingly, multiple data including HU treatment (Fig. 2E) and tests with retrotransposition-incompetent JM111 (Fig. 2C) and retrotransposition-competent sL1 (Fig. 2D) have suggested
Author contributions
K.Z., S.H., and X.F.Y. designed and supervised the project; K.Z., J.D., Y.P., P.L., S.W., Y.W., J.H., J.K., and W.Z. performed the experiments; K.Z., J.D., and S.H. analyzed the data; and K.Z., S.H., and X.F.Y. wrote the manuscript with the help of all authors.
Conflict of interest disclosure
All authors declare no conflict of interests.
Acknowledgments
We thank Dr. Yajuan Rui for technical assistance and Dr. Deborah McClellan for editorial assistance.
This work was supported in part by funding from the National Natural Science Foundation of China (81601363 to K.Z., 81401654 to J.D., and 81570002 to S.H.), China Postdoctoral Science Foundation (2017T100212 and 2017M610193 to K.Z.), the Chinese Ministry of Science and Technology (2012CB911100 and 2013ZX10001-005 to X.F.Y.), and a grant (yb201302 to K.Z.) from Norman Bethune Health Science Center
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