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.