| Popis: |
A significant number of known human genetic diseases is associated with nonsense mutations leading to the introduction of a premature termination codon into the coding sequence. A termination codon can be read through by its near-cognate tRNA (tRNA with two anticodon nucleotides base-pairing with a stop codon); potentially generating C-terminally extended protein variants. In yeast, UGA stop codon was described to be read through by tRNA-Trp and tRNA-Cys. Similar was observed for tRNA-Trp in human HEK293T cell line. The aim of this thesis was to investigate if human tRNA-Cys can act as a near-cognate tRNA in human HEK293T cell line. There are two isoacceptors which constitute the tRNA-Cys family, with ACA and GCA anticodon. There are 1 and 23 isodecoders to the ACA and GCA anticodons, respectively. Here, altogether as many as nine tRNA-Cys isodecoders (distinct in their sequence and with varying levels of expression) were tested for their ability to increase UGA readthrough in HEK293T using p2luci and pSGDluc dual-luciferase reporter vectors. In both p2luci and pSGDluc, we observed that at least one tRNA-Cys isodecoder, tRNA-Cys-GCA-4-1, is capable of significantly elevating the UGA readthrough levels when overexpressed in HEK293T. This indicates that similarly to yeast, tRNA-Cys is capable of... |
