ZFP91 And HNRNPU Regulate The Stability And Function Of METTL3

[Objective]RNA N~6-methyladenosine(m~6A)modification,a quite common post-transcriptional modification in eukaryotes,is installed by a complex consisting of multiple proteins such as METTL3,METTL14 and WTAP.Since this modification is a dynamically reversible process so it can be erased by FTO or ALKBH5.METTL3 is the only protein has methyltransferase activity in this complex,so it was targeted and researched in last year.At the same time,protein-protein interactions,key of biochemical reaction network,are significant in cell life activities.Therefore,this article mainly aimed to explore proteins that interact with METTL3 and in order to clarify the possible regulation between them and the biological functions.[Method]A large number of proteins that may interact with METTL3 were screened by Flag affinity purification technique combined with mass spectrometry analysis.Co-IP technique and immunofluorescence experiments were performed to investigate whether ZFP91 or HNRNPU interacted with METTL3 or not.Then we explored the biological functions of ZFP91 or HNRNPU after it interacted with METTL3.Western Blotting experiments were used to detect the expression of METTL3 affected by ZFP91.The half-time of METTL3 was measured after He La cell treated with CHX,and CQ or MG132 was used to investigate the mainly degradation pathway of METTL3.METTL3 ubiquitination level affected by ZFP91was detected by ubiquitination experiments.Realtime PCR or Western Blotting experiment was used to discover the expression level of METTL3,which might be influenced by HNRNPU.The RNA m~6A modification detection kit was used to evaluate the methylation level of METTL3.Me RIP experiment was used to test the quantity of TNFαwhich was methylation modified and the biology fuction affected by HNRNPU.The website SRAMP was used to predict the potential methylation site of TNFα.[Results]Flag affinity purification capture technique and mass spectrometry analysis were used to screen proteins that might interact with METTL3,and a total of 250 proteins were found finally.However,only ZFP91 or HNRNPU was selected for further investigation.Both ZFP91 and HNRNPU could interacte with METTL3and co-localized with METTL3 in nucleus.It has been found that ZFP91 could reduce the METTL3 protein level and shortened the half-time of METTL3 after He La cell was treated by CHX.After He La cells were treated by CQ or MG132,Western Blotting experiments were used to detect the protein level of METTL3,and we concluded that METTL3 degradated through proteasome pathway.Ubiquitination analysis showed that ZFP91 could promote the ubiquitination level of METTL3.Further investigation revealed that HNRNPU had no effect on the expression of METTL3,nor methyltransferase activity.However,HNRNPU might serve as a regulator and affected the methylation level of TNFα.[Conclusion]This study showed that ZFP91,an atypical E3 ubiquitin ligase,could affect the stability of METTL3 protein by promoting its ubiquitination level.HNRNPU mediated the m~6A methylation of TNFαby introducing METTL3 to enhance gene expression.