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\begin{document}

\title{Comparison of Xrn1 and Rat1 5'-3' exoribonucleases in budding yeast
supports the unique role of Xrn1 in co-translational mRNA decay}



\author[1]{Jose Perez-Ortin}%
\author[1]{A. Jordán-Pla}%
\author[2]{Y. Zhang}%
\author[1]{J. Moreno-García}%
\author[3]{L. Campos-Mata}%
\author[3]{Juana Diez}%
\author[2]{V. Pelechano}%
\author[1]{Jose Garcia-Martinez}%
\author[4]{Mordechai Choder}%
\affil[1]{Universitat de València}%
\affil[2]{Karolinska Institutet Institutionen for mikrobiologi tumor- och cellbiologi}%
\affil[3]{Universitat Pompeu Fabra}%
\affil[4]{Technion Israel Institute of Technology}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
The yeast \emph{Saccharomyces cerevisiae} and most eukaryotes carry two
5'-3' exoribonuclease paralogues that are very similar. In yeast, they
are called Xrn1, which shuttles between the nucleus and cytoplasm and
executes major cytoplasmic mRNA decay, and Rat1, which carries a strong
nuclear localization sequence (NLS) and localizes in the nucleus. Xrn1
is 40\% homologous to Rat1 but has an extra \textasciitilde{}500 amino
acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped
mRNAs during the last round of translation by ribosomes
``co-translational mRNA decay''. The division of labor between the two
enzymes is still enigmatic and can serve as a paradigm for division of
labor of many other paralogues. Here we show that Rat1 is capable of
functioning in cytoplasmic mRNA decay, provided that Rat1 remains
cytoplasmic due to its NLS disruption (cRat1). This indicates that the
actual segregation of the two paralogues plays roles in their specific
functions. However, segregation is not sufficient for fully
complementing Xrn1 function. Specifically, cRat1 can only partially
recover cell volume, mRNA stability, proliferation rate, 5'-3' decay
alterations that characterize \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}cells. In particular,
co-translational decay is only little complemented by cRat1. Adding the
Xrn1 C-terminal domain to Rat1 does not improve the phenotypes
indicating that lack of C-terminal is not the reason for the partial
complementation. Collectively, it seems that during evolution the two
paralogues acquire unique features that make the division of work
beneficial.%
\end{abstract}%



\sloppy


\textbf{Introduction}

In eukaryotes, 5'-3' decay plays a crucial role in controlling RNA
processing, quality, and quantity. 5'-3' exoribonucleases (XRNs) are
conserved in eukaryotes and play crucial roles in diverse aspects of
mRNA and non-coding (nc) RNA processing and degradation. XRNs recognize
RNAs with a 5' monophosphate (5'P) end arising from endonucleolytic or
exonucleolytic cleavage or decapping, and trim the RNA processively in
the 5'-3' direction (Nagarajan et al., 2013; Chang et al., 2011). The
molecular functions of XRNs have been mainly studied in the budding
yeast \emph{Saccharomyces cerevisiae} , which possesses two XRNs, a
mainly cytoplasmic exoribonuclease, Xrn1, and a nuclear exoribonuclease,
Xrn2 (also referred to as Rat1) (Amberg et al., 1992; Heyer et al.,
1995; Johnson, 1997). In yeast, Xrn1 is responsible for cytoplasmic
deadenylation-dependent mRNA 5'-3' exonucleolytic decay (Parker \& Song,
2004, Nagarajan et al., 2013). Indeed, the demonstrated activity as
5'-3' exoribonuclease (Larimer \& Stevens, 1990, Larimer et al., 1992)
is the reason for its Xrn1 (e\emph{x} o\emph{r} ibo\emph{n}
uclease\emph{1} ) name. Xrn1 5'-3' exonucleolytic activity on mRNAs is
fundamental for the decay of both correct and defective molecules,
provided that they have a 5'P end caused by either decapping or
endonucleolytic cleavage (Parker \& Song, 2004, Nagarajan et al., 2013).
Xrn1p can also hydrolyze NAD-capped mRNAs (deNADase activity) and,
afterwards, exonucleolytically degrade them processively (4). Its
enzymatic activity is highly processive and can even act
co-translationally on ribosome-associated mRNAs. In this context, Xrn1
trails the last translating ribosome (Pelechano et al, 2015) with which
it physically interacts specifically (Tesina et al., 2019). Xrn1 plays
additional roles in mRNA processing and quality control, rRNA
processing, tRNA quality and ncRNA decay (Parker \& Song, 2004;
Nagarajan et al., 2013; Langeberg et al., 2020; Sharma et al., 2022).

Both 5'-3' exoribonucleases are partially homologous (Nagarajan et al.,
2013; Kenna et al., 1993) and have been shown to be functionally
interchangeable, although they were thought to be restricted to specific
cellular compartments: Xrn1 in the cytoplasm and Rat1 in the nucleus
(Johnson, 1997). \emph{RAT1} is an essential gene, whereas \emph{XRN1}is
not (Johnson, 1997). Ectopic nuclear localization of Xrn1 by the
addition of an SV40 nuclear location signal (NLS) can rescue
\emph{rat1}lethality, whereas cytoplasmic Rat1, lacking its NLS, can
rescue\emph{xrn1 ski2} lethality (Johnson, 1997). \emph{SKI2} encodes a
co-factor of the 3'-5' RNA exosome, which is essential for its
cytoplasmic function (Johnson \& Kolodner, 1995). The enzymatic
mechanism of Rat1 is assumed to be processive and similar to that of
Xrn1 because it shares the active site and shows extensive conservation
around it (Nagarajan et al., 2013; Basu et al., 2021). Rat1 is also a
deNADing enzyme similar to Xrn1 (Sharma et al., 2022). Rat1 performs
important nuclear activities related to RNA metabolism, including rRNA
and snoRNA processing, as well as poly-A\textsuperscript{+} dependent
and independent mRNA transcription termination (Kim et al., 2004). The
existence of both nuclear (Rat1/Xrn2 family) and cytoplasmic (Xrn1
family) 5'-3' exoribonucleases is common in a variety of studied
eukaryotes (Chang et al., 2011; Han et al., 2023) and suggests that
functional interchangeability extends across species (reviewed in
Nagarajan et al., 2013).

Rat1 apparently lacks cytoplasmic functions (Johnson, 1997), whereas it
has been shown for Xrn1 that, albeit predominantly cytoplasmic (Johnson,
1997; Haimovich et al., 2013), it also plays important nuclear
functions, such as rRNA processing and degradation of defective RNAs
(Nagarajan et al., 2013). Xrn1 also acts as a general transcription
activator, forming a complex with other decay factors (Haimovich et al.,
2013; Medina et al., 2014). The proper cytoplasmic function of Xrn1p
requires its shuttling between the cytoplasm and nucleus (P\selectlanguage{ngerman}érez-Ortín \&
Chavez, 2022), which is made possible by the recently discovered
existence of two NLSs (Chattopadhyay et al., 2022). Budding yeast Xrn1
is a very long protein with 1528 amino acids compared to the shorter
Rat1 (1006 amino acids). The larger size of Xrn1 is due to an extended
C-terminal (Cterm) segment that is absent in Rat1 (Chang et al., 2012),
which is a basic domain that is not essential for most \emph{in
vivo}functions and is toxic when overexpressed (Page et al., 1998). This
feature and the relatively low homology between budding yeast Xrn1 and
Rat1 (40\% amino acid identity) pose the question of how these two
proteins can substitute one another when located in each other's cell
compartment (Johnson, 1997).

In this study, our goal was to gain insight into which of the \emph{in
vivo} activities of Xrn1 can be substituted with Rat1. We studied two
yeast strains that express \emph{RAT1} with defective NLS, the product
of which is cytoplasmic (cRat1). We found that cRat1 partially
complements many of the natural features of Xrn1, including
co-translational mRNA decay. This complementation is not improved by
adding Xrn1 Cterm to cRat1 despite the fact that this construction was
previously shown to complement other differences in cRat1 activity
(Blasco-Moreno et al., 2019). These results support the idea that both
5'\selectlanguage{english}-3' exonucleases have functionally redundant activities, but have
acquired some unique features, including their cellular localization,
that seem to optimize their function and possibly their cooperation. Our
results suggest that there are other structural disparities between the
two exonucleases, potentially involving distinct binding partners, or
the essential import of Xrn1 into the nucleus. These features are
crucial for the complete \emph{in vivo} functionality of
co-translational mRNA decay..

\textbf{Materials and Methods}

\textbf{Yeast strains and culture conditions}

Yeast cells were grown in YPD (1\% yeast extract, 2\% peptone, 2\%
glucose) at 30degC. Pre-cultures were grown overnight in 250 mL flasks and
shaken at 190 rpm. The next day, pre-cultures were diluted to
OD\textsubscript{600} = 0.05 and grown until an OD\textsubscript{600} of
\textasciitilde{}0.5 was reached. Cells were recovered by
centrifugation, flash-frozen in liquid nitrogen and stored at -20 oC
until needed for Genomic Run-on (GRO) or RNA extraction. Yeast strains
were transformed with centromeric plasmids YCpLac33 and pBBM3 following
a standard protocol (Gietz \& Schiestl, 2007). Yeast strains and
plasmids are listed in Supplementary Table S1.

For generation time (GT) estimations, 50 mL of yeast cultures were grown
in 250-mL flasks with shaking (190 rpm) at 30degC. Aliquots were taken
every 30 min in the exponential phase and their
OD\textsubscript{600}(from 0.05 to 0.7) were measured. The GT (in
minutes) in the exponential phase was calculated from growth curves (see
Figures 1 and S1).

The median values of cell volumes were calculated by a Coulter-Counter Z
series device (Beckman Coulter, USA). The absolute values in femtolitres
(fL) and relative values are shown in Figures 1 and S1.

\textbf{Genomic run-on}

Genomic run-on (GRO) was performed as described in (Garcia-Martinez et
al., 2004) and as modified in (Oliete-Calvo et al., 2018). Briefly, GRO
detects by macroarray hybridization, genome-wide, active elongating RNA
polymerase (RNA pol) II, whose density per gene is taken as a
measurement of its synthesis rate (SR). At the same time, the protocol
allows the mRNA amounts (RA) for all the genes to be measured. mRNA
half-lives (HLs) are calculated as RA/SR by assuming steady-state
conditions for the transcriptome. The total SR for a given yeast strain
was calculated as the sum of individual gene SRs. GRO datasets are
available at the Gene Expression Omnibus (GEO) with accession numbers:
GSE29519 and GSE198240 (reviewer access code: \emph{mbyjqgschrcfhib} ).

\textbf{mRNA half-life calculations}

In order to determine single gene mRNA HLs, transcriptional shut-off was
used following the addition of thiolutin up to 5 \selectlanguage{greek}µ\selectlanguage{english}g/mL. Samples were
collected at 0, 5, 12, 25, 45, 60 and 90 min after thiolutin addition.
Samples were analyzed by Northern blot using
specific\textsuperscript{32}P-probes as described elsewhere (Pelechano
\& P\selectlanguage{ngerman}érez-Ortín, 2008).

To determine the global mRNA HL, a dot-blot strategy was followed as
described in (García-Martínez et al., 2016). Known amounts of total RNA
were spotted onto a nylon membrane and hybridized with
a\textsuperscript{32}P-terminal labelled oligo
d(T)\textsubscript{40}probe. RNA samples were collected after thiolutin
addition at the same times as described for individual mRNAs. The
detailed protocol is described in (Benet et al., 2017).

\textbf{HT-5Pseq and 5'-Capseq protocols}

HT-5Pseq libraries were prepared as previously reported (Zhang \&
Pelechano, 2021). Briefly, 6 \selectlanguage{greek}μ\selectlanguage{english}g of DNA-free total RNA were directly
ligated with the RNA/RNA oligo containing UMI (RNA rP5\_RND oligo).
Ligated RNA was reverse-transcribed and primed with Illumina PE2
compatible oligos containing random hexamers and oligo-dT. RNA in
RNA/DNA hybrid was depleted by sodium hydroxide with 20-minute
incubation at 65\selectlanguage{ngerman}°C. Ribosomal RNAs were depleted using DSN
(duplex-specific nuclease) with the mixture of ribosomal DNA probes.
Samples were amplified by PCR and sequenced in an Illumina NextSeq 500
instrument using 60 sequencing cycles for read1 and 15 cycles for read
2.

For 5'Capseq, 5'capped mRNAs were captured as previously described
(Pelechano et al., 2016). Specifically, 10 \selectlanguage{greek}µ\selectlanguage{english}g total RNA were treated
with calf intestinal alkaline phosphatase (NEB, CIP) to remove 5'P
molecules (fragmented and non-capped). After purification, mRNA 5'caps
were removed by Cap-Clip (Biozyme), which resulted in 5'P molecules from
previously capped molecules. The following steps were the same as that
described for HT-5Pseq (see above) with variations only for skipping
removing ribosomal RNA. Datasets are available from GEO database:
GSE119114 for the 5Cap-seq data, GSE193992 (reviewer access
code:\emph{ahadwoocjrivtot} ) for HT-5Pseq data.

\textbf{Protein expression levels measurement by Western blotting}

To check for protein expression levels, yeast cells were transformed
with the corresponding plasmids and grown as specified in a previous
section. Two optical density (OD) units were harvested by
centrifugation. Total proteins were extracted from the equivalent number
of cells and loaded on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) to be separated according to their molecular
weight. Next samples were immunoblotted on a nitrocellulose membrane for
90 min at 100 V on ice, as previously described (Ishikawa et al., 1997).
Antibodies against FLAG (Sigma-Aldrich), PGK (Invitrogen) and Xrn1 (a
gift from A. Johnson) were used. Detection of proteins was performed
with an Amersham Imager 600.

\textbf{Bioinformatics procedures}

HT-5Pseq reads were trimmed with a 3'-sequencing adapter using cutadapt
V1.16 (http://gensoft.pasteur.fr/docs/cutadapt/1.6/index.html). The 8-nt
random barcodes on the 5~ \selectlanguage{english}ends of the reads were extracted and added to
the header of the fastq file as the UMI employing UMI-tools. 5`P reads
were mapped to \emph{S. cerevisiae} (SGD R64-1-1) using STAR 2.7.0 with
parameter --alignEndsType Extend5pOfRead1 to exclude soft-clipped bases
on the 5~ \selectlanguage{english}end. After removing PCR duplications using UMI-tools, analysis
of the 5~ \selectlanguage{english}ends positions was performed using the Fivepseq package
(Nersisyan et al., 2020, http://pelechanolab.com/software/fivepseq),
including the relative distance to start and stop codons. In particular,
the unique 5\selectlanguage{ngerman}´ mRNA reads within biological samples were summed and
subsequently normalized to reads per million (rpm). Standalone
normalized average density plots around genomic features were calculated
with R and Python software ngs.plot v2.61 (Schen et al., 2014) using
indexed alignment files as inputs and the internal SacCer3 database
annotation as reference. The statistical robustness parameter, which
filters out 0.5\% of the genes with the most extreme (high and low)
count values, was applied to all calculations.

5'Cap reads were processed as described for HT-5Pseq reads. In general,
5'Cap reads were trimmed using a 3'-sequencing adapter and the extracted
8-nt random barcodes were used as UMI. The 5'Cap reads were mapped
to\emph{S. cerevisiae} (SGD R64-1-1) using STAR 2.7.0. PCR duplicates
were removed using means of UMI-tools.

Heatmaps and the accompanying average metaplots displaying HT-5Pseq
alongside the 5'Capseq datasets were generated using the bamCoverage,
computeMatrix and plotHeatmap functions from the deepTools2 package
(Ramírez et al., 2026). The transcription start site (TSS) and polyA
site (pA) annotations were taken from (Xu et al., 2009). Spearman
correlation values and statistical test result asterisks were inserted
into ggplot2-generated plots using the ggpubr package
(\emph{https://rpkgs.datanovia.com/ggpubr}).

\textbf{Results}

\textbf{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 \selectlanguage{english}\emph{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
the\selectlanguage{english}\emph{xrn1[?]} strain, including the cell volume, proliferation rate
and mRNA stabilities. We transformed the wild-type and \selectlanguage{english}\emph{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 \emph{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 (\emph{RPL25} ) and
high (\emph{ACT1} ) mRNA stability in an \selectlanguage{english}\emph{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 \selectlanguage{english}\emph{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 \selectlanguage{english}\emph{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 \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}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 \emph{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
(Garc\selectlanguage{ngerman}ía-Martínez et al., 2016). Alternatively, or in addition, it is
possible that 5'\selectlanguage{english}-3' mRNA decay \emph{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 \selectlanguage{english}\emph{xrn1[?]} were
more compensated after cRat1 addition (Supplementary Figure S2).
However, the analysis of the transcriptome-wide differential expression
in \selectlanguage{english}\emph{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.

\selectlanguage{english}\textbf{Cytoplasmic Rat1 can function co-translationally in mRNA 5'-3'
exonuclease, albeit inefficiently}

A quintessential function of Xrn1 is to degrade decapped mRNA during its
last round of translation. This helps terminating translation and
contributes to half-lives of many mRNAs (see Introduction). We next
investigated whether cRat1 could perform ribosome-associated
co-translational decay. For this end, we measured the presence of
5'-Phosphate-containing mRNAs genome wide by 5'P degradome RNA
sequencing (HT-5Pseq) on both sets of four strains: a) the set of
strains transformed with a plasmid containing the Rat1-\selectlanguage{english}[?]NLS version
(cRat1) without FLAG epitopes or with the empty vector (Figure 2); and
b) the set of strains (wild-type and \selectlanguage{english}\emph{xrn1[?]} ) transformed, or not,
with cRat1-3xFLAG (Supplementary Figure S3). We decided to use the FLAG
tagged cRat1 as we could be sure about its equal level in all the
studied strains (Fig. S3), whereas using the untagged strain was made to
make sure that Rat1 function was not compromised.

The wild-type HT-5Pseq metagene profile was characterized by a
3-nucleotide periodicity pattern and a prominent peak of HT-5Pseq reads
at -17 nt from the STOP codon (Pelechano et al, 2015). HT-5Pseq measures
the distance between the 5\selectlanguage{english}'\selectlanguage{english}-3\selectlanguage{english}'exonuclease and trailing ribosome during
co-translational mRNA decay. We have previously shown that the distance
between the 5'P of the mRNA undergoing degradation and the first base of
the termination codon is 17 nt at the A site of the trailing ribosome
(Pelechano et al, 2015; Zhang \& Pelechano, 2021). This distance
reflects the steric protection offered by the ribosome to the \emph{in
vivo} trimming action of Xrn1p. In contrast to wild-type the -17 nt peak
was completely lost in \selectlanguage{english}\emph{xrn1[?]} and, in the 5'UTR, reads accumulated
in the zone immediately upstream of the AUG codon (Figure 2A). cRat1
partially restored the wild-type metagene profile to show intermediate
levels for the 5'-upstream and -17 nt peaks (Fig. 2 left and right
panels, respectively). An approach to evaluate genuine ribosome
occupancy was to determine how well the coding frame was protected from
5' to 3' decay in relation to the other two frames. The ratio between
the protected frame and the other frames was designated as the ``frame
protection index'' (FPI) (Pelechano et al, 2015). This index measures
the effectiveness of single-nucleotide coupling between nuclease
activity and ribosome position. As expected, the FPI was compromised
in\selectlanguage{english}\emph{xrn1[?]} cells (Figure 2B). The expression of cRat1 in
the\selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}cells only partially recovered the FPI (from 0.79
in\selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}increased to 0.82 in cRat1, compared to value 1 for the
wild-type FPI; see Figure 2B). This finding suggests that Rat1 is
inefficient, closely following the ribosome position during
co-translational mRNA decay when placed in the cytoplasm. This might
reflect either differences in the enzyme localization, configuration or
processivity.

Given that HT-5Pseq reads accumulate at the 3' (close to the STOP codon)
in wild-type cells and shifted to 5' (close to the AUG codon)
in\emph{xrn1[?]} , we reasoned that the proportion of reads in these two
regions could serve as a proxy for the processivity of the 5'-3' decay
level once decay has started, taken as either a genome-wide average or a
per-gene index. To calculate this 3' \emph{vs.} 5' proportion for the
wild-type, \emph{xrn1[?]} and cRat1 strains, we first made metagene
profiles by spanning the entire gene body (Figure 2C), and then compared
the number of reads in the last 20\% \emph{vs} . the first 20\% of the
average gene body region. These 3'/5' indices showed that expressing
cRat1 in \emph{xrn1[?]} was half as efficient as the wild-type in 5'-3'
decay (Figure 2D). We next used the per-gene 3'/5' indices to
investigate whether sets of functionally related genes were skewed
towards any end of the range. A functional classification analysis of
the wild-type revealed that the 250 genes with the highest values in the
3'/5' index (high 5'-3' degradation: Figure, 2E upper panel) were
enriched in Gene Ontology (GO, Biological Process) terms related to cell
cycle and stress response (not shown), whereas the 250 genes with the
lowest values (low 5'-3' degradation: Figure 2E, lower panel) were
enriched in GOs related to mitochondria and glucose metabolism, although
with low significance (not shown). Next, we generated metagene plots for
specific gene sets and observed that while the overall shapes of
profiles slightly differed for the various groups, the proportions of
reads between samples remained approximately the same in all four yeast
strains (Supplementary Figure S3B). This result indicates that the lack
of Xrn1 (\emph{xrn1[?]} ) and its substitution by cRat1 have
transcriptome-wide effects. The heatmaps of the individual genes ordered
by 5'UTR length (Figure 2F and Supplementary Figure S4) show that the
average metagene plots (Figure 2A/C) represent truly uniform behavior
for most yeast mRNAs, with two stronger regions at the 5': being most
reads associated with the TSS (see Supplementary Figure S4) and ordered
according to 5'UTR length, and a minor one located around the AUG codon
as we have previously described for multiple species (Huch et al.,
2023). This distribution is clearly observed in the summary average
count metagene plots (top panels in Figures 2F and S4). The TSS peak
detected by HT-5Pseq was particularly clear for \emph{xrn1[?],} and
slightly less so for cRat1. The position of this peak corresponded to
the canonical capped TSS full-length molecules (as measured by 5CapSeq,
the rightmost lane in Figures 2F and S4). Altogether, these results
suggest that in \emph{xrn1[?]} , and to a lesser extent in cRat1, a large
fraction of the 5'P-detected molecules corresponds to decapped
full-length mRNAs.

FPI reflects single-nucleotide coupling between co-translational decay
and the ribosome position along the coding region, while the 3'/5' ratio
reflects the processivity of the 5'-3' exonuclease activity and ribosome
protection along mRNA. The 3'/5' index is mainly influenced by
translation initiation and termination, which are the two regions where
ribosomes tend to pause the most. Because in the wild-type pausing at
the stop codon lasts longer than pausing at the start codon, the general
3'/5' ratio is expected to be higher than 1. A slow 5'-3' decay results
in a 3'/5' index lower than 1 (negative Log\textsubscript{2} ratio in
Figure 2D). Nevertheless, the translation machinery can also protect
mRNA from 5'-3' decay, perhaps because the cap is protected (e.g., by
eIF4F). Indeed, highly translated mRNAs are less prone to global
co-translational decay (Pelechano et al, 2015) and 5'-3' decay. In fact,
the 3'/5' index shows a statistically significant inverse correlation
with mRNA stability and individual translation rate in wild-type cells
(TLRi data from For\selectlanguage{ngerman}és-Martos et al., 2021) (Figure 2G and Supplemental
Figure S5). These correlations support the notion that the higher the
ribosome density (related to TLRi), the greater the protection against
5'\selectlanguage{english}-3' decay, and the higher the mRNA stability. This effect depends on
Xrn1 because the negative correlation disappears in \selectlanguage{english}\emph{xrn1[?]} , which
is consistent with Xrn1 being the major 5'\selectlanguage{english}-3' exonuclease (Haimovich et
al., 2013; Parker, 2012). The substitution of Xrn1 for cRat1, however,
cannot restore ribosome density dependence (Figure S5), consistent with
the poor capacity of Rat1 to complement the function of Xrn1 in
co-translational decay.

\textbf{The C-terminus of Xrn1 does not complement the defect in
co-translational decay that characterizes the cRat1 function}

The inability of cRat1 to fully complement \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}phenotypes,
including the turnover of cytoplasmic mRNAs, suggests that cRat1 does
not recognize the uncapped mRNAs, unlike Xrn1 does, or it is not
localized close enough to the 5' ends of cytoplasmic mRNAs. In a
previous study, we demonstrated that cRat1 was unable to properly
regulate the translation of a subset of membrane protein-coding mRNAs
because it lacked the C-terminal domain of Xrn1 (Blasco-Moreno et al.,
2019). Therefore, we reasoned that the large unstructured domain of
Xrn1, which seems to be necessary for interaction with the 5'UTR of
these specific mRNAs, might also be necessary to achieve wild-type
global 5'-3' exonuclease activity and co-translational 5'-decay levels.

Therefore, to investigate the possible function of Xrn1 Cterm, we used
it to create a fusion protein, cRat1-Cterm-3xFLAG, and expressed it in
an \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}background. The protein levels were somewhat lower than
those of regular cRat1-3xFLAG, probably because of the much larger
fusion protein size (Supplementary Figure S6A). It is important to note
that cRat1-Cterm with a FLAG epitope had no noticeable effect on the
5'-3' exonuclease activity of cRat1 (see Figures 2 and S3). Both the
cell growth and co-translational decay defects of \emph{xrn1[?]} were
compensated only partially by cRat1-Cterm to a similar extent as by
regular cRat1 (see the cRat1-Cterm profiles in Figures 2A, C),
suggesting that Xrn1 performs specific functions that cannot be replaced
with Rat1, even when their main structural difference is minimized using
a Rat1-Xrn1-Cterm chimera.

\textbf{Discussion}

During evolution, organisms have accumulated paralogues. As this is a
widespread phenomenon, a question was raised regarding the functions of
paralogues, do they serve as backups, or each paralogue has acquired a
unique function? Here we focus on two paralogues, Rat1 functions mainly,
if not exclusively, in the nucleus, whereas Xrn1 shuttles between the
nucleus and cytoplasm and accumulates in the cytoplasm. As these
paralogues are active in different compartments, the issue of their
potential capacity is especially interesting. It was proposed that the
two paralogues are fully replaceable, and their distinct functions are
attributed to their cellular localization (Johnson, 1997). Xrn1 is a
highly conserved and exceptionally large protein (175 kDa in yeast) with
multiple biological functions in many eukaryotes. Many of these
functions can be attributed to its enzymatic 5'-3' exonuclease activity
which acts on several types of RNAs. Rat1 is smaller (116 kDa in yeast)
and also functions in several nuclear RNAs decay pathways, including
those linked to RNA pol II-coupled mRNA transcriptional termination (Kim
et al., 2004). Both Xrn1 and Rat1 substrates are single-stranded
unstructured 5'Phosphate end with lengths of at least four nucleotides
(Nagarajan et al., 2013; Basu et al., 2021). Many of Xrn1 functions
require only two-thirds of the molecule, which is homologous to Rat1.
The last Xrn1 C-terminal third (CTD) is apparently unstructured, less
conserved, and not important for some functions (Bashkirov et al.,
1995). CTD is necessary for Xrn1 interaction with eIF4G to regulate the
translation of certain membrane-protein-encoding mRNAs (Blasco-Moreno et
al., 2019). It also serves as an interaction platform for other proteins
in higher eukaryotes (Braun et al., 2012). Being unstructured, it is
likely involved in Xrn1 capacity to reside in liquid-liquid
phase-separated droplets (Currie et al., 2020). To perform nuclear
functions, Xrn1 shuttles between the cytoplasm and nucleus (Haimovich et
al., 2013; Medina et al., 2014; P\selectlanguage{ngerman}érez-Ortín \& Chavez, 2022). Nuclear
import depends on two NLSs whereas export to the cytoplasm is proposed
to be mediated by its ability to bind or ``imprint'' mRNAs
(Chattopadhyay et al., 2022). Shuttling and mRNA imprinting have been
shown to affect both mRNAs synthesis and decay (Chattopadhyay et al.,
2022). Thus, although these two paralogues are very similar and both
have highly conserved domains that carry 5'\selectlanguage{english}-3' exonuclease activity,
their cellular localization is different. This raised the question
whether this difference in localization is the main reason for their
disparate functions. This question was previously addressed by A.
Johnson (1997), who conclusdedthat they are functionally
interchangeable. Because we have gained more information about these
paralogues since 1997, we decided to reevaluate this conclusion. The
above question is important for understanding the functions of these two
paralogues. Moreover, addressing this issue can also serve as a paradigm
for other paralogues with identical enzymatic activity.

To this end, we investigated whether the cytoplasmic function of Xrn1
could be performed by its nuclear counterpart Rat1. We found that a
cytoplasmic version of Rat1 (cRat1) can only partially complement the
5'\selectlanguage{english}-3' exonuclease activity of the missing Xrn1 in the cytoplasm. Even
fusing the Xrn1 CTD to the C-terminus of cRat1 (cRat1-Cterm), did not
improve cRat1 performance. Specifically, although cRat1 could mostly
recover cell volume, growth rate and global mRNA decay, it fails to
fully recover co-translational mRNA decay. Previously, we found that
deletion of XRN1 resulted in defective transcription, which was
attributed to both direct and indirect effects (Haimovich et al., 2013;
Medina et al., 2014; Chattopadhyay et al., 2022). Direct effects should
be the result of the loss of the direct function of Xrn1 in
transcription activation (Xrn1 is a component of the pre-initiation
complex), whereas indirect effects are probably the consequence of slow
growth of the strain. Given the observation that Rat1 recovers the
growth defect of xrn1\selectlanguage{english}? cell, the cRat1-mediated increase in
transcription of these \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}cells might be due to the growth
rate effect. Interestingly, cRat1 could slightly, but significantly,
increase transcription in wild-type cells. cRat1 might compete with Xrn1
on binding the substrates. This possible competition could provide a
plausible explanation for this transcriptional increase in wild-type
cells, as cRat1 could increase the amount of free Xrn1, capable of
importing to the nucleus and activating transcription. In addition, it
is possible that mRNA decay \emph{per se} enhances transcription (e.g.,
by supplying more nucleotides).

In the absence of \emph{XRN1} , cRat1 can provide some mRNA buffering
activity (Figure 1A). Hence, it seems that a partial mRNA buffering can
be accomplished without Xrn1 and without its specific shuttling feature.
Interestingly, we found that compensation introduced by cRat1 is
proportional to the defect caused by the absence of Xrn1 (Figure S2).
Maybe, the observation that decapping rate is one of the limiting
activities in mRNA decay (Parker 2012) is relevant for this correlation.
We propose that mRNA degradation \emph{per se} can provide some
buffering, probably together with other pathways and partners that act
in concert or in parallel. In particular, our results suggest that the
transcription activation capacity of Xrn1 is not absolutely required for
buffering (assuming that Rat1 does not have this function) However,
since this buffering is partial, it is clear that mRNA decay is not
sufficient to provide full buffering. Maybe the transcription activation
and the specific shuttling features of Xrn1 are required for efficient
buffering.

The effect of cRat1 expression on the start of 5'-3' mRNA decay after
decapping (HT-5Pseq profiles and the 3'/5' index) of \selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}cells
is partial. Using heatmaps of the HT-5Pseq data, we noticed that in
wild-type cells, the peak at the TSS was clearly less strong than in
the\selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}cells (Figures 2F and S4), which suggests that Xrn1 is
located in the vicinity of the RNA 5' end, and is ready to act as soon
as the cap is removed. cRat1 did not significantly improve
the\selectlanguage{greek}\emph{xrn1Δ} \selectlanguage{english}pattern. The 3 nt degradation pattern inside the ORFs in
a\selectlanguage{greek}\emph{xnr1Δ} \selectlanguage{english}is lost. Taking the relative FPI value of this mutant
lacking any cytoplasmic 5'-3' exoribonuclease as a bottom reference
(0.79) and the wild-type value as 1, the cRat1 strain only increases the
FPI value by 14\% (up to 0.82) indicating that cRat1 is not a good
substitute for Xrn1 closely following the ribosome position during
co-translational mRNA decay. Given that Cterm of Xrn1 does not improve
the function of cRat1 we conclude that the N-terminal two-thirds of Xrn1
contain all the determinants needed for 5'-3'co-translational decay.
This idea is supported by the fact that the Xrn1 interacts with
ribosomes via the 1-772 amino acids region and no interaction is
detected with its Cterm domain (Tesina et al., 2019). The reason why
cRat1 can hardly replace Xrn1 in co-translational decay remains to be
determined. It is possible that the imprinting capacity of Xrn1
(Chattopadhyay et al., 2022) plays some role. The association of the
imprinted Xrn1 with mRNA also in the cytoplasm may localize Xrn1 near
the 5' end of the mRNA, and upon decapping it is available immediately
for degradation. We note that the expression level of cRat1 is probably
lower than the cytoplasmic level of Xrn1 in the wild type. However,
given that it has been shown that the cytoplasmic level of Xrn1 is not
limiting for 5'-3' mRNA decay (Chattopadhyay et al., 2022) we consider
that this is not the reason of the differences found between cRat1 and
wild type cells.

\emph{XRN1} and \emph{RAT1} are paralogous genes that are not derived
from the whole-genome duplication (Wolfe, 2015) but from an older
small-scale duplication (Fares et al., 2013). They show intermediate
homology divergence and are present in most eukaryotes from yeast to
humans (Parker \& Song, 2004), suggesting a long time for sequence
divergence. However, they can reciprocally rescue each other's lethal
phenotypes in yeast (Johnson, 1997). Here we confirm that their
exonuclease activities are interchangeable. Nevertheless, it seems that
the specialization of the two 5'-3' exonucleases makes them different in
terms of the interactive partners they bind to, perhaps affected by the
presence or absence of Xrn1 Cterm. However, the addition of Xrn1-Cterm
to Rat1 was insufficient to make it fully interchangeable with Xrn1.
Therefore, it is important to note that, their subcellular distribution
and dynamics are critical for their differential activities. Rat1 is
predominantly nuclear whereas Xrn1 is largely cytoplasmic (Sharma et
al., 2022; Johnson, 1997; Haimovich et al., 2013). Rat1 can substitute
for Xrn1 when its NLS is deleted and becomes cytoplasmic (Sharma et al.,
2022, this paper) whereas Xrn1 can only complement the \emph{rat1}mutant
when it overexpressed from a multicopy plasmid and is fused to a strong
SV40 large-T-antigen NLS (Johnson, 1997). Xrn1, moreover, has the
capacity to shuttle. Thus, the number of nuclear and cytoplasmic 5'-3'
exonuclease molecules and the capacity to shuttle determine the
functional difference between these two exonucleases. These features
should have appeared early during eukaryotic evolution and made it
impossible to completely substitute one protein for the other, despite
their similar and partially interchangeable exonuclease activities.

\textbf{Funding}

This work was funded with grants from: the Spanish
MCIN/AEI/10.13039/501100011033 {[}PID2020-112853GB-C31{]} to J.E.P-O;
the Swedish Foundation's Starting Grant (Ragnar S\selectlanguage{ngerman}öderberg Foundation);
the Swedish Research Council {[}VR 2020-01480, 2021-06112 and
2019-02335{]}; a Wallenberg Academy Fellowship {[}2021.0167{]}; Vinnova
(2020-03620), Karolinska Institutet (SciLifeLab Fellowship, SFO and KI
funds) to V.P. and the Israel Science Foundation (ISF) for M.C., Y.Z. is
funded by a fellowship from the China Scholarship Council. The
computational analysis was partially performed on the resources provided
by the Swedish National Infrastructure for Computing (SNIC) through the
Uppsala Multidisciplinary Center for Advanced Computational Science
(UPPMAX), partially funded by the Swedish Research Council with grant
agreement no. 2018-05973.

\textbf{Author Contributions}

A. Jordán-Pla: validation, formal analysis. Y. Zhang: Investigation.
Formal analysis; J. Moreno-García: Investigation; L. de Campos-Mata:
Investigation; M. Choder: Conceptualization, Manuscript writing; J.
Díez: Conceptualization; V. Pelechano: Conceptualization, Funding; J.
García-Martínez: Investigation, Formal analysis; J.E. Pérez-Ortín: Study
design, Investigation, Formal analysis, Manuscript writing, Funding.

\textbf{Acknowledgements}

We acknowledge A. Johnson for the gift of the Xrn1 antibody.

\textbf{Conflict of interest}

The authors declare no conflict of interest



\textbf{Figure legends}

\selectlanguage{english}\textbf{Figure 1.- The cytoplasmic version of the Rat1 protein (cRat1)
restores mostly, but not totally, the main phenotypes of a
\emph{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
\textless{} 0.001 level (***).

\selectlanguage{english}\textbf{Figure 2.- The cytoplasmic version of Rat1 protein (cRat1)
partially restores the wild-type HT-5Pseq profile in a
\emph{xrn[?]1}strain. 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 \selectlanguage{english}\emph{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 \textless{} 0.001; **** =
P \textless{} 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 log\textsubscript{2} 3'/5' index values for the wt, xrn1\selectlanguage{greek}Δ \selectlanguage{english}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 log\textsubscript{2} 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 For\selectlanguage{ngerman}és-Martos et al., 2021).

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