Cytoplasmic Rat1 partially replaces Xrn1 in global phenotypes
Rat1 derivative lacking the NLS (cRat1) which mis-localizes to the
cytoplasm, was complements some of the cytoplasmic functions of Xrn1,
including the defective growth phenotype of the xrn1∆ strain
(Johnson, 1997; Blasco-Moreno et al., 2019), growth in the presence of
benomyl (Johnson, 1997) and the stability of some viral mRNAs
(Blasco-Moreno et al., 2019). In the present study, we investigated
whether cRat1 complemented the global physiological phenotypes of thexrn1∆ strain, including the cell volume, proliferation rate and
mRNA stabilities. We transformed the wild-type and xrn1∆ strains
with a centromeric plasmid containing the cRat1 gene (pBBM3) using an
empty vector (YCpLac33) as a control. Notably, the wild-type strain
expressing cRat1 also expressed the endogenous wild-type (including NLS)
nuclear Rat1 version (Blasco-Moreno et al., 2019). In this study, these
yeast strains are referred to as cRat1 and wt+cRat1 for simplification
(Supplementary Table S1). Using this series of four strains we analyzed
global poly(A) mRNA stability by transcription shutoff with thiolutin
and dot-blot hybridization of time-course samples. We found that cRat1
reverses most of the global increase in mRNA stability caused by Xrn1
depletion (1.38x vs. 2.43x, regarding the wild-type considered as
1x; Figure 1A). On the other hand, the addition of cRat1 to a wild-type
strain with natural Xrn1 in the cytoplasm did not alter poly(A) decay
(0.98x), which suggests a minor role of cRat1 in mRNA decay when
endogenous Xrn1 is also present. Finally, cRat1 partially restored the
generation time and cell size of the wild-type (Figure 1A).
To verify cRat1 expression, we constructed and analyzed a series of four
strains in which the cRat1 protein has a 3xFLAG epitope in its
C-terminal part, and validated its expression by western blotting in
yeast cells (Figure S1A). The 3xFLAG epitope was necessary to observe
the cRat1 expression because 1xFLAG version does not allow detection
(Supplementary Figure S1A) but partially affects the strain
proliferation rate (Figure S1D). cRat1 without the FLAG epitope, but
expressed from the same construct (see Supplementary Table S1) grew
similarly to the wild-type (Figure S1D). In these strains we measured
mRNA half-lives in two individual mRNAs with medium (RPL25 ) and
high (ACT1 ) mRNA stability in an xrn1∆ strain, and found
that cRat1-3xFLAG restored wild-type stabilities (Figure S1B). To
further assess the effect of cRat1 on mRNA stability, we analyzed global
poly(A) decay. This analysis showed that cRat1-3xFLAG partially restored
wild-type global poly(A) mRNA stabilities (Figure S1C) similar to the
cRat1 version (Figure 1A). The generation time and cell size were closer
to those of the wild type but not fully recovered (Figures S1C and S1D).
as in the case of the strains without the FLAG epitope (Figure 1A).
Interestingly, the non-cytoplasmic version of Rat1 with FLAG epitopes
did not rescue the growth defect of the xrn1∆ strain
(Supplementary Figure S1D). We conclude that cRat1 with or without the
FLAG epitope behaves similarly in the phenotypes analyzed, although the
FLAG epitope has a certain detrimental effect. The actual cell volumes,
mRNA global stabilities and generation times for both sets of four
strains (Figures 1 and S1) are not quantitatively identical but they
qualitatively confirm that cRat1 partially restores global phenotypes in
an xrn1∆ background.
As cells lacking Xrn1 displayed a strong global down-regulation of RNA
pol II-based transcriptional activity (Haimovich et al., 2013), we next
examined whether cRat1 would mitigate the effects on global RNA pol II
synthesis rates. By means of Genomic run-on (GRO), we found that mRNA
synthesis rate in xrn1Δ was 0.36x that of wild type, whereas
addition of cRat1 partially compensated this drop (0.63xFigure 1B). This
partial compensation suggests that the decrease in RNA pol II synthesis
rates in the xrn1∆ mutants was due not only to the absence of
Xrn1 as a transcription activator (Medina et al, 2014), but also to the
indirect effect of its lower growth rate on synthesis rates
(García-Martínez et al., 2016). Alternatively, or in addition, it is
possible that 5’→3’ mRNA decay per se is important for
transcription. Interestingly, the global synthesis rates of cRat1 added
to a wild-type strain slightly, but significantly, increased (1.15x;
Figure 1B) without having a noticeable effect on global mRNA stabilities
(Figure 1A).
The compensation in mRNA synthesis rates and stabilities in a cRat1
strain showed a strong bias in individual mRNA synthesis rates and
stabilities: the more affected mRNAs in either one in xrn1∆ were
more compensated after cRat1 addition (Supplementary Figure S2).
However, the analysis of the transcriptome-wide differential expression
in xrn1∆ and the wild-type strains supplemented with cRat1 did
not show strong biases in either synthesis rates or mRNA stabilities
towards specific gene categories (Supplementary Table S2). This
indicates that cRat1 does not have a significant bias in the mRNAs of
genes belonging to functional groups, but is mostly related to the
actual synthesis and decay rates of mRNAs.