The Mechanisms Of RIG-Ⅰ As A RNA Helicase Regulating The Differentiation And Proliferation Of Granulocyte

Retinoic acid inducible gene I(RIG-Ⅰ),also known as DEAD box protein 58(DDX58),is a gene that up-regulated during the differentiation and maturation of acute promyelocytic leukemia(APL)cell line NB4 induced by all-trans retinoic acid(ATRA).RIG-Ⅰ is a conservative RNA helicase,which consists of N-terminal caspase recruitment domain(CARD),RNA helicase domain(helicase)and C-terminal regulatory domain(CTD).Numerous studies have shown that RIG-Ⅰ plays a crucial role in antiviral innate immunity response,and defined as an intracellular RNA receptor.It can identify a variety of intracellular viral RNA,especially 5’ppp-ds RNA produced during their replication,by CTD domain.Subsequently,RIG-Ⅰ interacts with mitochondrial antiviral signaling protein(MAVS)through its CARD domain,resulting in activating IRF3 and NFκB and induces type I interferons.However,RIG-Ⅰ has firstly reported as a gene up-regulated in NB4 cell line during ATRA-induced differentiation at a virus-free state.It is closely related to the proliferation and differentiation of granulocyte.Knockout of Rig-i in mice leads to abnormal phenotypes associated to hematopoietic differentiation or immune response,such as myeloproliferative neoplasms(MPN),granulocyte hyperplasia,hepatosplenomegaly infiltration,colitis,and even embryonic death.At present,a small number of studies have showed that RIG-Ⅰ can interact with STAT1,Src and other proteins to restrain leukemic stemness and cell proliferation and promote cell differentiation,which might interprets the mechanism of RIG-Ⅰ regulating hematopoiesis to some extent.Expert virtual specific 5’ppp-ds RNAs,RIG-Ⅰ has also been found that can also recognize poly-U/UC or U/A enriched UTR sequence without phosphate groups.It reported that RIG-Ⅰ could bind to endogenous RNA to regulate immunity or cell proliferation.However,the molecular mechanisms underlying RNA helicase RIG-Ⅰ-mediated regulating the differentiation of granulocyte is unclear.In the current study,we took protein-RNA interaction as the breakthrough point and explored the mechanisms of RIG-Ⅰ as a RNA helicase regulating the differentiation and proliferation of granulocyte with NB4 and RIG-Ⅰ inducible U937 cell line.First,we performed RIP-Seq analysis and found that RIG-Ⅰ could interact with a series of endogenous RNA including m RNA,lnc RNA,mi RNA,and sno RNA in the process of inducing granulocytic differentiation.Second,we focused on the interaction between RIG-Ⅰ and TRIM25 m RNA,which plays a critical role in the RIG-Ⅰ-mediated antiviral immune response.We confirmed repeatedly that RIG-Ⅰ could bind to the m RNA of E3 ubiquitin/ISG15 ligase TRIM25 and increase its stability through helicase and CTD through experiments such as RIP,RNA pulldown and kinetic experiments,result in upregulation of TRIM25 protein.Besides,we also found that RIG-Ⅰ could activate STAT1 by CARD to promote the transcriptional expression of TRIM25.Moreover,RIG-Ⅰ could also up-regulate the expression of other genes in the ISGylation pathway and the overall ISGylation level.Finally,we found that knockdown of TRIM25 or ISG15 could inhibit the differentiation of granulocyte to a certain extent.Co-IP/MS data showed that TRIM25 could bind and modify proteins closely related to cell differentiation and proliferation,such as EZH2,Cyclin D1,and PCNA.Therefore,we make a conclusion that RIG-Ⅰ,as a multi-domain protein,can bind endogenous RNAs by helicase activity in the process of granulocytic differentiation.RIG-Ⅰ could up-regulate TRIM25 and activate ISGylation pathway though both RNA binding and protein signal transduction.Finally,the ISGylation modifications of hematopoietic associated proteins regulate the differentiation and proliferation of granulocyte Our results have elucidated that the molecular mechanism that RIG-Ⅰ could act as a RNA helicase to regulate the differentiation and proliferation of granulocyte,thus providing a new perspective for studying the ligands and biological functions of RIG-Ⅰ.