Structural And Functional Studies Of Novel M~5C RNA Binding Proteins YPS And YBX1

RNA 5-methylcytosine(m5C)is a modification with relatively high abundance at messenger RNA(m RNA),however,research on this modification has long been focused on t RNAs and r RNAs.With the development of sequencing technology and mass spectrometry technology,the mystery of the role of RNA 5-methylcytosine modification on messenger RNA is gradually being unveiled.Along with the high-resolution single-base mapping of RNA 5-methylcytosine modifications,the distribution of RNA 5-methylcytosine on m RNA and related proteins including reader,writer and eraser were discovered.The reader protein for RNA5-methylcytosine reported so far is ALYREF,which is mainly located in the nucleus.However,it is unknown whether the RNA 5-methylcytosine on m RNA in the cytoplasm has its unique reader protein.Finding and identifying novel RNA5-methylcytosine-modified binding proteins will help to explore the physiological functions of RNA 5-methylcytosine.In this study,we identified a human Y-box binding protein 1(YBX1),which belongs to the Y-box family of RNA binding proteins and binds RNA through a conserved cold shock domain.In vitro binding assay verified the ability of YBX1 to bind RNA 5-methylcytosine,and it has higher affinity for RNA with5-methylcytosine than non-methylated RNA.Further,the crystal structures of YBX1 complexes with 5-methylcytosine modified RNA and non-methylated modified RNA revealed the molecular basis of recognition.First,we performed the mutations at binding sites W65,F74,F85,H87,K118.Then,the result of the mutations and m5 C RNA shows the hydrophobic interactions is important.The hydrogen bond between K118 and m5 C also improves the binding affinity.In addition,we collaboratively explored the physiological functions of Ypsilon Schachtel(YPS),a homologous protein of YBX1 using the Drosophila melanogaster.YBX1 from human could compensate the development deficiency in YPS deletion flies,suggesting two homologs are functionally conserved.At the same time,Flag-tag pull down and co-immnoprecipitation showed that YPS preferentially bind to m RNA containing RNA 5-methylcytosine modification in vivo.Sequence alignment showed YPS share the same amino acids with YBX1 around the 5-methylcytosine recognition site.The YPS mutation flies fail to bind RNA 5-methylcytosine are dominant negative,and display abnormal germline stem cell development.To summarise,our works identify a novel RNA 5-methylcytosine binding protein,which provides a powerful molecular tool to study the function of RNA5-methylcytosine modification.The crystal structure of YBX1 in complex with RNA 5-methylcytosine reveals the molecular mechanism of epigenetic modification.Disrupting the RNA 5-methylcytosine recognition by YPS will influence germline stem cell development.Moreover,YBX1 is overexpressed in a variety of cancers,the findings of this study provide new insights for the design of potential drugs in the future.In addition,we can develop new RNA editing tool based on our findings.