Structure And Function Of The Arabidopsis Terminal Uridylyltransferases URT1

At present,it is known that more than one hundred post-transcriptional modifications affect the fate of RNAs,which can affect the function,location,stability and are closely related to various human diseases.Currently known widely modified RNA includes rRNAs,tRNAs,snRNAs,miRNAs and other non-coding RNAs.Yet,RNA modifications are not restricted to non-coding RNAs and mRNA in archaeal bacteria,bacteria and eukaryotes.The addition of nucleotides to the 3’ end of coding RNAs and non-coding RNAs in eukarytes is an important post-transcripitional modification,which is divided into adenylation,uridylation,cytidylation,and guanylation according to the type of nucleotide addeds.The function of adenylation is to decaying RNAs and activiting translation,cytidylation and guanylation have rarely been studied so far.But the research on uridylation has made breakthrough progress in recent years.The untemplated addition of uridines,which is called uridylation,helps regulating gene expression,such as triggering RNAs degradation,promoting RNAs processing or maintaining RNAs stability.The enzymes that help to catalyze uridylation are terminal uridylyltransferases(TUTases),and belong to the DNA polymerase β superfamily which mainly composed of DNA polymerase β nucleotide transferase domain(NTD)and poly(A)polymerase-associated domain.The NTD is located at the N-terminus of proteins and contains three conserved aspartic acid(Asp)or glutamic acid(Glu)to chelate divalent metal ions to complete the catalysis,so we can call it the catalytic domain(CAT domain).PAP contains a type Ⅱ-nucleotide recognition motif(NRM),in which the histidine(His)is involved in UTP recognition,and PAP can also be called the center domain(CD domain).From lower to higger organisms,the structure of transferases tends to be complicated.In addition to NTD and PAP,multipartite domain for instance zinc finfers have evolved to mediating protein-protein interactions or protein-RNA binding.HEN1 SUPRESSOR 1(HESO1)is identified as uridylyltransferases for most demethylated miRNAs in Arabidopsis.And subsequent studies found that when HESO1 was absent there are another transferase,UTP:RNA URIDYLYLTRANSFERASE 1(URT1),for uridylating miRNAs.That is,URT1,as the second highest influence TUTase in Arabidopsis,can add uridines tail to different forms of RNAs substrate with HESO1 in vivo.However,they prefer miRNA substrates with distinct 3’ nucleotides in vitro.HESO1 prefers 3’U,while URT1 prefers 3’A.And they act on different size variants of the same miRNAs in vivo.Researches revealed URT1 and HESO1 act sequentially on some miRNAs,with URT1 mono-uridylating the miRNAs followed by their further uridylation by HESO1.It is not only URT1 has been reported to have a preference for the 3’terminal nucleotide of RNAs.In 2018,we reported how the Drosophila TUTase Tailor selectively catalyze the 3’G modification of RNA substrates on NAR.In order to further study the molecular mechanism of URT1/HESO1 regulation of Arabidopsis RNA metabolism.Firstly,we resolved the crystal structure of URT1,URT1-UTP and URT1-AAAU.This series of structures is the first report of TUTases in plant so far.Combining with functional studies in vitro and sequence alignment analysis,we found that URT1 is conserved in evolution,and it has a similar UTP recognition mechanism as Cidl in Schizosaccharomyces pome in which serine and lysine residues mainly interact with UTP by hydrogen-bonding,tyrosine residue stabilize UTP by π-π stacking and H714 selectively recognize UTP.R327 and N347 are two key residues lead to preference for 3’G in Tailor,and the efficiency of enzyme activity is greatly reduced while R327A or N347A.Nevertheless,the corresponding R531 or N552 mutation in URT1 does not significantly affect the efficiency of enzyme activity.So we speculate the preference mechanism may not be the same as Tailor.Finding the key amino acids that affect the prefrrence of URT1 requires further experimrntal verification.Fianlly,the overall structure changes and the catalytic domain folds towards the center after URT1 binds to RNA.In addition,R531 deflects greatly and forms hydrogen interaction with D700 in URT1-AAAU.This phenomenon has not been found in homologous proteins.