Mechanism Research Of Aicardi-Goutières Syndrome Protein MDA5 Suppresses Reverse Transcription Element LINE-1

Background and objectives:RNA sensing plays an important role in innate immune activation inside human cells.It senses the presence of non-self RNA(viral RNA,for instance)and activates downstream pathways,leading to the expression of interferons(IFNs).Subsequently,IFN triggers the expression of interferon-stimulated genes(ISGs),many products of which function as suppressors against exogenous pathogens.One of the well-studied RNA sensors is melanoma differentiation-associated protein 5(MDA5).MDA5 is not only a promoter of increased IFN production,but also an effector of IFN activation(i.e.,IFN promotes MDA5 expression).This is likely to indicate that MDA5 may have some innate post-immune activation function in addition to sensing RNA.Therefore,it would be easy to imagine that the malfunction of MDA5 can trigger innate immune dysregulation.Mutations in MDA5 have consistently been associated with an autoimmune disease termed Aicardi-Goutières syndrome(AGS).Type 1 long interspersed elements,or LINE-1,are the only active retroelements in human cells.The retrotransposition process of LINE-1 can trigger the activation of the innate immune system and has been proposed to play a role in the development of several autoimmune diseases,including Aicardi-Goutières syndrome(AGS).In contrast,all known AGS-associated proteins,except MDA5,have been reported to affect LINE-1 activity.Given the complex relationships between MDA5 and AGS,LINE-1 and innate immune activation,and IFN and MDA5,we speculated that MDA5 might be an effective inhibitor of LINE-1.Methods:1.LINE-1 retrotransposition assay was performed,and the effect of MDA5 on LINE-1 activity was evaluated by flow cytometry and Western-blot assay.Reverse verification was performed by specific si RNA silencing MDA5 assay.2.Real-time quantitative polymerase chain reaction(q RT-PCR)was performed to confirm that MDA5 overexpression could increase the production of endogenous interferon(IFN),and the m RNA level of endogenous ISGs was detected by q RT-PCR.MDA5 mutants deficient in innate immune activation and RNA binding were constructed through polymerase chain reaction(PCR),reverse transcription,RNA extraction,plasmid ligation and other experiments,then LINE-1 retrotransposition assay was performed to evaluate the effects of IFN pathway and RNA binding of MDA5 on LINE-1 activity regulation.3.The main pathway of LINE-1 regulation by MDA5 was evaluated at protein level,RNA level and promoter level by Western-blot,PCR assay and promoter activity assay based on firefly luciferase expression respectively.4.Co-immunoprecipitation assay was used to identify the functional regions of MDA5 which plays the key role in LINE-1 activity regulation.Results:In this paper,the biological functions of MDA5 are systematically studied and the following results are obtained:1.MDA5 can effectively inhibit the activity of the reverse transcription of LINE-1In a recent study,LINE-1 ribonucleoprotein particles(RNP)were found to act as endogenous triggers that activate the innate immune system via the RNA sensing pathway,providing direct evidence of a relationship between retrotransponders,innate immune activation,and IFN-based autoimmune diseases.Interestingly,one of the RNA sensors involved in the above pathway is MDA5.Since all known AGSrelated proteins except MDA5 have been reported to affect LINE-1 activity,we speculated that MDA5 might also be an inhibitor of LINE-1.In this study,it was found that exogenous expression of MDA5 in HEK293 T cells could significantly inhibit LINE-1 activity,whereas endogenous MDA5 was silenced and LINE-1activity was significantly increased,indicating that endogenous MDA5 also regulated LINE-1 replication.Consistent results were also obtained in He La cell lines,further confirming the fact that MDA5 is indeed a LINE-1 inhibitor.2.IFN elevation,though unessential,contributes to MDA5-mediated LINE-1suppressionMDA5 is an initiator of the RNA sensing pathway that triggers the activation of the natural immune system.Experimental results showed that increased exogenous IFNβ protein in HEK293 T cells significantly inhibited LINE-1 replication.Most likely,ISG expression is induced by intracellular pathways.Further experiments showed that MDA5 hardly increased endogenous IFNβ expression under similar level of LINE-1 inhibition,while exogenous IFNβ was easily detected,suggesting that IFN activation may not be the key mechanism of MDA5-mediated LINE-1 inhibition.On the other hand,MDA5 mutants with obvious deficiency in natural immune activation ability,and mutants which reduced the effectiveness of MDA5 binding RNA,were only slightly less capable of inhibiting LINE-1 than the wild-type.Therefore,although increased IFN and subsequent ISG protein levels contribute to MDA5-mediated LINE-1 inhibition,our results suggest that a more effective pathway may play a major role in MDA5-mediated LINE-1 inhibition.3.The inhibition of LINE-1 by MDA5 is mainly achieved through the inhibition of promoter activityThe mechanism of MDA5 regulating LINE-1 is still unclear.Our experiment further confirmed that MDA5 inhibits LINE-1 mainly by inhibiting promoter activity through verification at protein level,RNA level and promoter level.4.The 2CARD region is important for MDA5-mediated LINE-1 suppressionThe total length of MDA5 contains 1025 amino acids,which are composed of Nterminal 2CARD domain,DEx D/H helicase domain,and C-terminal domain(CTD).In order to clarify the key region of MDA5-mediated LINE-1 inhibition,we truncated the MDA5 functional region continuously from N-terminal to C-terminal,and the results showed that truncations containing 2CARD region retained an obvious ability to inhibit LINE-1,and could regulate the 5’-UTR by combining with 5’-UTR.Conversely,truncation without 2CARD region cannot bind to 5’-UTR and almost completely lose the ability to inhibit LINE-1.Thus,the key role of 2CARD region in MDA5’s inhibition of LINE-1 was clarified.Conclusions:To sum up,this paper not only confirmed the inhibition of AGS-related protein MDA5 on LINE-1 activity for the first time,but also explained the mechanism of MDA5 on LINE-1 inhibition from various aspects,and clarified the main pathway and functional areas of MDA5 on LINE-1 regulation.It provides a solid molecular basis for exploring the complex relationships between MDA5 and AGS,LINE-1 and innate immune activation,IFN and MDA5,and may provide new molecular mechanisms and targets for the prevention and treatment of autoimmune diseases such as AGS.