Figure legends
Figure 1.- The cytoplasmic version of the Rat1 protein (cRat1) restores mostly, but not totally, the main phenotypes of a xrn1∆strain. A) Generation times (GT), cell volumes and global poly(A) mRNA stability were determined as described in the Materials and Methods section for the four strains. Values were relativized to those of the wild-type (wt) strain (transformed with the empty plasmid YCpLac33), which was taken as 1.00. As a result, the wild-type (wt) values lack standard deviation (SD). The experiments were repeated three times and averaged. The actual values for wt were 80±9 min for GT, 65±2 fL for cell volume and 59±6 min for poly(A) mRNA stability. B) The synthesis rates (SR) for all RNA pol II genes (in arbitrary units, a.u.) were calculated by GRO, as described in the Materials and Methods. The distribution of all values and their medians are shown in the box and whiskers representation. All comparisons are significant at the < 0.001 level (***).
Figure 2.- The cytoplasmic version of Rat1 protein (cRat1) partially restores the wild-type HT-5Pseq profile in a xrn∆1strain. The C–terminal region of Xrn1 does not improve the performance of cRat1 . A) HT-5Pseq high-resolution plots showing the profiles of wild-type (wt) and xrn1∆ strains transformed with the empty YCpLac33 plasmid or its version (pBBM3), including the truncated cytoplasmic version of Rat1 (cRat1) without the nuclear localization sequence (NLS) or the same construct fused to the C-terminal domain of Xrn1 (cRat1-Cterm). The metagene analysis for 5’P read coverage in relation to the open reading frame (ORF) start (left) and stop codons (right) are shown in the samples described above. Three biological replicates of each experiment were merged for each sample. B) Relative frame protection index (FPI) of the four strains analyzed in A). The median FPI for the wt was taken as 1. *** = P < 0.001; **** = P < 0.0001. C) Average metagene plots of normalized HT-5Pseq read counts around the protein-coding genes. Gene body read coverage is represented as a percentage of the total length of the ORF, whereas the flanking regions around the START and STOP codons represent the real distances from the reference points (represented as base pair length). Shadowed vertical areas highlight the 20% length of both transcribed region ends used for the calculation of the 3’/5’ index. D) Violin plot of log2 3’/5’ index values for the wt, xrn1Δ and cRat1 strains. E) Average metagene plots of 250 genes with the highest 3’/5’ index values (top panel) or 250 genes with the lowest (bottom panel) in wt. F) Heatmaps of HT-5Pseq and 5’Capseq data for all the individual protein-coding genes aligned by their START codon and ordered, from top to bottom, by increasing the 5’UTR length. The corresponding summary average count metagene plots are shown at the top of each heatmap. G) Violin plots of the log2 frame protection index (FPI) (left) or 3’/5’ index (right) values in wt for all protein-coding genes, ribosomal proteins (RP) and genes with high or low individual translation rates (TLRi; as described in Forés-Martos et al., 2021).