Study of the cellular component-dependent functions of Xrn1

The main cytoplasmic mRNA decay pathway in yeast uses the 5'-3' exonuclease Xrn1 (Parker and Song, Nat Struct Mol Biol. 2004 ). This protein shuttles from the cytoplasm to the nucleus, where it has a role as transcription factor (Haimovich et al. Cell 2013). In this work, we find that most of the global phenotypes of an xrn1 mutant are partially complemented by a cytoplasmic version of the paralogous 5'-3'exonuclease Rat1 (cRat1) indicating that this 5'-3'-exonuclease has a similar enzymatic capacity as Xrn1. The lack of a cytoplasmatic 5'-3'-exoribonuclease is the cause of the physiological defects of an xrn1 mutant. The capacity of cRat1 to perform co-translational decay is, however, very limited. The comparison with the strain having a NLS1?-NLS2?-Xrn1 version shows that it is slightly deficient in 5'?3'-co-translational decay but much more efficient than cRat1. In both strains, cRat1 and -Xrn1-NLS1&2, the lack of nuclear Xrn1 has a very minor influence on cell growth. Overall design: We performed HT-5PSeq experiments on 2 sets of strains to measure if cytoplasmic Rat1 was able to perform ribosome-associated co-translational decay : set #1: BY4741 (wt) and xrn1? transformed, or not, with Rat1-?NLS-3xFlag. This set includes BY4741, xrn1?, BY4741 + Rat1-?NLS-3xFlag (wt+cRat1), xrn1?+Rat1-?NLS-3xFlag (xrn1+cRat1). Set #2) BY4741 (wt) and xrn1? transformed with a plasmid containing the Rat1-?NLS version without the FLAG epitopes, the same Rat1-?NLS plus the Xrn1 C-termainal domain or with the empty vector. These three plasmids are: YCpLac33 (URA3, CEN4ARS1, empty vector), pBBM3 (YCpLac33+Rat1?NLS) and pLCM9 (YCpLac33+ Rat1?NLS-Xrn1-Cterm-3xFLAG). No transformation of pLCM9 in BY4741 was done. Additionally we analyzed Set #3 of strains to explore whether co-translational decay was dependent on nucleo-cytoplasmic shuttling of Xrn1. We performed HT-5PSeq on Xrn1 with a version lacking one or both NLS sequences (Xrn1-NLS1&2). This set includes Xrn1-?NLS1, Xrn1-?NLS2, Xrn1-?NLS1&NLS2. All the three sets of strains were done in duplicate or in triplicate samples for each strain grown in YPD to middle exponential phase (0.5 OD600).