IRES-mediated Translation Of Pot1 N-terminal Deleted Protein

Shelterin complex consisting of TRF1,TRF2,RAP1,TPP1 and Pot1 is important for telomere protection and telomere regulation.The protection of temomeres(Potl)protein is the only telomeric single-stranded binding protein in shelterin with two N-terminal oligonucleotide-/oligosaccharide-binding(OB)fold domains.Recent researches show mutations were frequently in Pot1 gene regions encoding the OB-fold domains in chronic lymphocytic leukemia and familial cutaneous malignant melanoma,and with these mutations Potl could not bind telomeric single-stranded DNA,which suggested that changed Potl station in chromosome end was associated with tumorigenesis.Our experiments show Pot1 had an unknown IRES sequence in the first OB-fold domain of mRNA encoding sequence,which contributed to the translation of a full-length Potl protein and a N-terminal OB-fold deleted Potl fragment.In addition,a two-tag wide type Pot1 plasmid was constructed to express in various cells,the full-length Potl and N-terminal deleted Pot1 fragment could also be detected.After excluding antibody,vector and tag factors,more experiment methods and approaches were used,such as a inner pre-terminated mutant,an open reading frame shifted mutant,an expressed protein fusion and a bi-cistron vector,to demonstrate directly that Potl had an IRES sequence in the N-terminal 2-132 amino acids(the first OB-fold domain),and this IRES sequence could mediate independently the translation of its down-stream protein or peptide.Further,cell cycle synchronization show the activity of the IRES sequence in Potl mRNA was regulated by cell cycles.The expression ratio of the full-length Pot1 protein and the N-terminal deleted Potl fragment was significantly improved in the cell cycle G2/M,which may be implicated in the binding of Potl and telomeric single-stranded DNA.Our researches demonstrated firstly that the mRNA of Potl had an IRES sequence,and it could regulate the expression ratio of the full-length Pot1 protein and N-terminal deleted Potl fragment.this was a novel mechnism for Pot1 binding telomeric single-stranded DNA in chromosome end.As the discovery of the association between Potl binding telomeric single-stranded DNA and human tumorigenesis,our researches will play important roles in the study of IRES regulaton mechanism of Pot1 mRNA in the unknown function of tumors in the future.