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).