Cloning And Subcellular Localization Of Polyadenylation Factor Of Arabidopsis Thaliana

Polyadenylation is a process of cutting at the cleavage site of pre-mRNA 3' ends,and then adding poly(A)tail to form mature mRNA.Polyadenylation is a key step and an important hub for plant gene expression regulation.Polyadenylation requires lots of poly(A)factors,such as cleavage and polyadenylation specific factor(CPSF),cleavage stimulation factor(CstF),cleavage factor Im(CF-Im),cleavage factor CFIIm(CF-IIm),symplekin,poly(A)polymerase(PAP)and poly(A)binding protein(PABN).Currently research of pre-mRNA polyadenylation is mainly focused on animals and fungi.The poly(A)factors involved in this process is mainly located in the nucleus and cytoplasm.In the higher plant Arabidopsis thaliana,however,the poly(A)factors are only seen in the nucleus acorrding to the present report.In order to systematically explore the function and subcellular location of poly(A)factors in Arabidopsis thaliana,43 Arabidopsis poly(A)factors were selected for cloning and subcellular localization in this study.Multiple sequence alignment and evolutionary analysis of 13 poly(A)factors were carried out between species and intraspecific.The results showed that the two subunits of CFIm25 were conserved,indicating the similar function among various species.Furthermore,the 13 poly(A)factors of the mice were very similar to those of human.Poly(A)factors in animals,plants and yeast are conservative in evolution,suggesting polyadenylation of pre-mRNA is a conservative gene expression regulation mechanism in the biological world.We successfully cloned 32 of the 43 poly(A)factors,and constructed recombinant plasmid pCAMBIA3301-ACT2-Poly(A)factor.The results of tobacco transient transformation showed that 15 poly(A)factors,CPSF73(AT1G61010),CstF64(AT1G71800),ESP1(AT1G73840),CFIm25,(At4g25550,At4g29820),PCFS(At4g04885),CLPS(AT3G04680),PABN(At5g65260,At5g51120),PAP(At2g25850,At4g32850),Symplekin(AT1G27595,AT5G27590)and GLD-2 like(At2G45620,AT2G40520)were scuccesfuly tansformed.After observed with a fluorescence microscope,we found that poly(A)factors were successfully expressed in the epidermal cells of tobacco leaves.CFIm25 and GLD-2 like(coded by At2G45620 and AT2G40520)are expressed in both the cytoplasm and nucleus,and CFIm25 is mainly expressed in the cytoplasm,suggesting that the factors are not only participated in the pre-mRNA processing,but also play a role in the cytoplasm.The factor symplekin coded by AT5G27590 is expressed only in the cytoplasm,suggesting that it may not be required in Arabidopsis polyadenylation,and it might play an important role in the cytoplasm,which need further research to prove.The factors CPSF73(I)(AT1G61010),CstF64(AT1G71800),ESP1(AT1G73840),PCFS(At4g04885),CLPS(AT3G04680),PABN(At5g65260 and At5g51120),PAP(At2g25850 and At4g32850),Symplekin(AT1G27595),are only expressed in the nucleus.Finally,we use Agrobacterium tumefaciens mediated bud soaking method to transform Arabidopsis thaliana with recombinant plasmid.Western blot were further used to confirm that 11 poly(A)genes overexpressed in Arabidopsis,which is CPSF73(AT1G61010),ESP1(AT1G73840),CFIm25(At4g25550,At4g29820),PCFS(At4g04885),PABN(At5g51120),Symplekin(AT1G27595,AT1G27570)and GLD-2 like(AT3G56320,AT2G40520,AT2G39740).Meanwhile,we also observed that some poly(A)overexpression lines exhibited early flowering phenotype.To sum up,the present work lays a foundation for further study of the function of poly(A)factors in Arabidopsis thaliana,and clarifying the mechanism of Arabidopsis polyadenylation.Moreover,it provides a theoretical basis for the research of human diseases thatrelated to polyadenylation.