Screening Of Interacting Proteins Of RNA M6A Methyltransferase MTA And MTB From Arabidopsis And Rice

Among the epigenetic modification of eukaryotes,N6-methyladenosine(m6A)is the most common epigenetic modification in mRNA.It is widely present in various of RNA molecules and performs different biological functions.The m6A modification was first discovered in mammalian cells,and is dynamically decomposed by m6A methyltransferase,recognition factors,and demethylase.The m6A methyltransferase complex mainly has three major components,METTL3,METTL14 and WTAP.In mammalian cells,METTL3 has catalytic activity,METTL14 and WTAP do not have catalytic activity,but as a regulatory subunit,WTAP interacts highly with METTL3 and METTL14.In order to verify whether the MTA and MTB proteins(homologous proteins of the human genes METTL3 and METTL14)interact with each other in Arabidopsis,and to find possible unknown proteins in the m6A methyltransferase complex,we constructed a GFP-tagged MTA and MTB gene overexpression vectors,and obtained transgenic lines with MTA and MTB overexpression in Arabidopsis thaliana wild type background.Finally,39 and 49 MTA and MTB gene positive transgenic lines were obtained,respectively.Laser confocal microscopy observed the expression of GFP protein,and found that 6 and 7 single lines of MTA and MTB overexpression transgenic plants,respectively,can emit green fluorescence under blue excitation light.Further analysis revealed that MTA is not only distributed in the nucleus,but also in the cell membrane,which shows that MTA may have more biological functions.In order to verify the expression level of the target genes,we randomly selected from overexpressing transgenic lines that can stimulate green fluorescence,found that the MTA gene expression was up-regulated 2404 times compared to the wild type,and the MTB gene expression was up-regulated 366 times.Western Blot detected with GFP antibody showed that MTA and MTB fusion GFP protein was translated correctly.Combining immunoprecipitation and mass spectrometry to identify the various proteins contained in the complex,MTB protein was identified in both repeats of MTA Co-IP mass spectrometry results,and MTA was also identified in both repeats of MTB mass spectrometry results.This is consistent with previous studies,indicating that the experimental results are reliable.Excluding the control proteins,Co-IP mass spectrometry identified 50 MTA interaction proteins and 57 MTB interaction proteins.In rice,the orthologous genes of MTA OsMTA2(LOC_Os02g45110),MTB orthologs OsMTA1(LOC_Os01g16180).OsMTA3(LOC_Os03g05420),OsMTA4(LOC_Os10g31030)were found through evolutionary analysis in rice.With the exception of OsMTA2,the other three genes have all been cloned,an eGFP-tagged overexpression vector has been constructed,and genetic transformation has been completed.After identification,37 OsMTA1 overexpressing rice lines,40 OsMTA3 overexpressing rice lines and 39 OsMTA4 overexpressing rice lines were obtained.In summary,the corresponding transgenic materials were obtained by constructing m6A methyltransferase overexpression vectors in rice and Arabidopsis thaliana,and found that MTA has an unknown location in the cell and some new proteins interacted with MTA and MTB.The creation of related materials and the identification of interaction proteins have laid a good foundation for further exploring the function of plant m6A methyltransferase.