Fig. 2: Some mutant Tail-GFP molecules are localized constitutively in the nucleus.

Tail (amino acid 351 until 521 of the XRN1 sequence) was fused to GFP and was expressed under ADH1 promoter and terminator. Nup49-RFP (a nucleoporin) was used as a nuclear marker. a During optimal proliferation, Tail is mainly cytoplasmic. b Tail-GFP shuttles between cytoplasm and nucleus. Tail-GFP localization was examined by the import assay, as in Fig. 1c. Photos were taken after 2 h at 37 °C. c-e Mutant Tail-GFP molecules that are constitutively localized to the nucleus. The Tail sequence of Tail-GFP was randomly mutagenized by PCR mutagenesis protocol and introduced into WT strain by transformation. Distinct colonies were allowed to proliferate in 96 wells. The cellular localization of the mutants was automatically scanned by fluorescence microscopy using a robot. Shown are some of the mutants that were localized to the nucleus in optimally proliferating cells. See Fig. S2d for positions of the mutations. f Tail^S454P nuclear import is mediated by NLS1. NLS1 of Tail^S454P was mutagenized (resulting in Tail^{S454P, ΔNLS1}), and the mutant was analysed as in c. g Localization of mutant Tail-GFP in temperature-sensitive (ts) nuclear-import mutant. A shuttling assay, using cells that express nup49-313 and co-expressed xrn1^S454P and RPB7-RFP (as a positive control), was performed as described previously²⁵. h Quantification of the shuttling assay. The nuclear/whole-cell ratio of the mean fluorescence intensity was determined by ImageJ, as described in Methods. Each box represents the 25th to 75th percentile of values, with the median noted by the horizontal bar. Whiskers terminate at maxima/minima or a distance of 1.5 times the IQR away from the upper/lower quartile, whichever is closer. n = >100 cells examined over 3 independent experiments. P-value was calculated by Wilcoxon rank sum test. ** - p < 0.01. ns – not significant.