The Study On Translation Initiation Regulation In Sperm Cell Of Arabidopsis Thaliana

Translation is the process during which the genetic information carried by mRNA is converted into the protein by ribosome and includes three steps:translation initiation,translation extension,and translation termination.Translation initiation is the critical step to regulate the efficiency of protein synthesis and the protein abundance,during which the start codon AUG is found by 43S preinitiation complex(PIC)through scanning mechanism and the protein synthesis is initiated.In animal cells,previous studies have shown that the translation initiation has the highest efficiency and is not affected by the translation initiation factor(eIF1)under the optimal context(Kozak sequence),while in the non-optimal context,initiation efficiency is low and negatively regulated by the translation initiation factor(eIF1).Unlike other cells,the sperm cell in Angiosperms does not exist independently,but is surrounded by the vegetative cell.It has not been studied whether this special structure makes sperm cell have a different mechanism on translation initiation than normal cells.In this study,using Arabidopsis thaliana,we investigated the following four questions:(1)whether does the mRNA expressed in sperm cell have a special nucleotide composition near the start codon AUG?(2),whether is the efficiency of translation initiation in sperm cell affected by nucleotide sequences near the start codon AUG?If so,is it affected by nucleotide sequences before or after AUG?(3),do the "hot spots" in nucleotide sequences near AUG regulate the efficiency of translation initiation?(4),does the translation initiation factor eIF1 affect the efficiency of translation initiation in sperm cell?The results of this study are as follows.(1)The nucleotide composition of sperm cell mRNA near the start codon AUG is not specific,compared with that in normal cells.By analyzing 1000 mRNA sequences expressed in sperm cell and on chromosome 1,we found that the upstream and downstream 15-18 nucleotides at AUG exhibited a distribution biased to nucleotide A.The frequency was about 30%.The nucleotide U occurred at a frequency slightly lower than A,and the nucleotide C occurred at the lowest frequency of about 18%.Among them,the nucleotides of A at the position-1 to-4 occurred at a frequency of 42%?50%,and the nucleotide of G at the position+4 is present at a frequency of about 57%.(2)The translation initiation efficiency in sperm cell was significantly affected by the upstream nucleotide sequence of the starting codon AUG,but slightly affected by the downstream nucleotide sequence of AUG Through expressing the fusion genes with different nucleotide background in sperm cell,we observed that the efficiency of the translation initiation under the optimal upstream background was 9 times that under the poorest background.Meanwhile,the efficiency of translation initiation under the optimal 15 downstream nucleotides at AUG was twice that under the poorest downstream nucleotide background.These results first indicate that the nucleotide sequences near the start codon AUG can significantly affect the efficiency of translation initiation in sperm cell,and the effect is mainly attributed to the upstream sequence of AUG.(3)Hot spots of nucleotide played a key role in the efficiency of translation initiation.Under the poorest nucleotide background with the nucleotides of A at the position-1 to-4,the translation init iation efficiency was increased significant ly,which was 79%of the efficiency under the optimal upstream background.This indicates that the hot spots at the position-1 to-4 play an important role in regulating translation initiation.Moreover,the translation initiation efficiency in a normal nucleotide background with the As at the position-1 to-4 was four times that in the same background with the poorest nucleotides at the position-1 to-4.This further shows that the hot spots at the position-1 to-4 play an important role in translation initiation regulation.(4)Translation initiation factor eIF1 did not affect the efficiency of translation initiation in sperm cell.Through the single sperm cell RT-PCR,we found that ateIF1 was expressed in sperm cell.Under the poorest nucleotide background,translation initiation efficiency was not affected by either overexpression of ateIFl or knockdown of ateIF1.The results suggest that the translation initiation factor eIF1 do not affect the efficiency of translation initiation in sperm cell.These results reveal for the first time that the upstream nucleotide sequences at AUG in the sperm cell of A.thaliana can significantly affect the efficiency of translation initiation,and this effect is mainly attributed to the nucleotides at the position-1 to-4.Furthermore,the translation initiation factor eIF1 do not participate in the translation initiation regulation in sperm cell.These results provide new insights on understanding the development of sperm cell at the cytological level.