Mechanism On Selection Of MiRNA-mediated M Rna Cleavage Or Translation Repression In Arabidopsis

Micro RNAs(miRNAs)are a class of 20-to 24-nucleotide(nt)long endogenous noncoding singe-stranded RNAs that are present in eukaryotes.In plants,miRNAs negatively regulate the expression of target genes and play important roles in cell proliferation and differentiation,growth and development,and responses to environmental stresses.Plant miRNAs mainly mediate m RNA cleavage and translation repression.The cleavage of target m RNAs leads to their degradion as well as the decreasing of protein levels,while translation suppression only affects the protein levels.Mechanisms underpinning the action of plant miRNAs are still not well understood,especially with respect to the influencing factors on the determination of m RNA cleavage or translation repression.It has been reported that the previously known degree of miRNA-target complementarity and the binding sites of target genes(5?-UTR,coding region or 3?-UTR)are not the determining factors of the selection of the two modes of action of plant miRNAs.This study focuses on the mechanism on selection of miRNA-mediated m RNA cleavage or translation repression in Arabidopsis.By constructing fusion vectors carrying a constitutive promoter(TUA2or PTB2)and an artificial miRNA(ami RSUL or ami RBAK1),we obtained a series of transgenic homozygous lines with different miRNA expression levels,and detected the expression levels of target m RNAs and proteins to analyze the modes of action of miRNAs.We also detected the translation efficiency of target m RNAs on ribosomes,and analyzed the data of m RNA sequencing.Several conclusions are drawn as follows.Firstly,ami RSUL mediates the cleavage of target m RNAs,and translation suppression is undetectable.With the increasing expression level of ami RSUL,the abundance of SUL m RNA decreases,and the protein level declines as well.Secondly,as the expression level of ami RSUL increases,it shows an elavated level of SUL m RNA distributing on polyribosomes,suggesting that the translation efficiency of SUL m RNA is positively regulated in the cells,so as to compensate the suppression of SUL and photosynthesis.Thirdly,ami RBAK1 mediates translation suppression,and the level of full-length m RNA of BAK1 were comparable to that in wild type.Fourthly,in our reporter system,the abundance of miRNAs is uncorrelated to the selection of the two modes of action of plant miRNAs.Fifthly,BAK1 m RNA is significantly enriched on membrane-bound polyribosomes(MBPs),but it shows no siginificant enrichment of other target m RNAs,which were known to involve in the miRNA-mediated translation repression,on MBPs,implying that the enrichment of target m RNAs on MBPs is not necessary to the mode selection.Our research explores the mechanism on the selection of miRNA-mediate m RNA cleavage or translation repression in Arabidopsis,aiming to stimulate further research on the modes of action of plant miRNAs.Especially,the reporter lines only participating in translation suppression are valuable materials for the genetic screening of mutants that affect translation repression.