The C-terminus of Xrn1 does not complement the defect in co-translational decay that characterizes the cRat1 function
The inability of cRat1 to fully complement xrn1Δ phenotypes, including the turnover of cytoplasmic mRNAs, suggests that cRat1 does not recognize the uncapped mRNAs, unlike Xrn1 does, or it is not localized close enough to the 5’ ends of cytoplasmic mRNAs. In a previous study, we demonstrated that cRat1 was unable to properly regulate the translation of a subset of membrane protein-coding mRNAs because it lacked the C-terminal domain of Xrn1 (Blasco-Moreno et al., 2019). Therefore, we reasoned that the large unstructured domain of Xrn1, which seems to be necessary for interaction with the 5’UTR of these specific mRNAs, might also be necessary to achieve wild-type global 5’→3’ exonuclease activity and co-translational 5’-decay levels.
Therefore, to investigate the possible function of Xrn1 Cterm, we used it to create a fusion protein, cRat1-Cterm-3xFLAG, and expressed it in an xrn1Δ background. The protein levels were somewhat lower than those of regular cRat1-3xFLAG, probably because of the much larger fusion protein size (Supplementary Figure S6A). It is important to note that cRat1-Cterm with a FLAG epitope had no noticeable effect on the 5’→3’ exonuclease activity of cRat1 (see Figures 2 and S3). Both the cell growth and co-translational decay defects of xrn1∆ were compensated only partially by cRat1-Cterm to a similar extent as by regular cRat1 (see the cRat1-Cterm profiles in Figures 2A, C), suggesting that Xrn1 performs specific functions that cannot be replaced with Rat1, even when their main structural difference is minimized using a Rat1-Xrn1-Cterm chimera.