Regulated translational bypass of stop codons in yeast.

Abstract

Stop codons are used to signal the ribosome to terminate the decoding of an mRNA template. Recent studies on translation termination in the yeast Saccharomyces cerevisiae have not only enabled the identification of the key components of the termination machinery, but have also revealed several regulatory mechanisms that might enable the controlled synthesis of C-terminally extended polypeptides via stop-codon readthrough. These include both genetic and epigenetic mechanisms. Rather than being a translation 'error', stop-codon readthrough can have important effects on other cellular processes such as mRNA degradation and, in some cases, can confer a beneficial phenotype to the cell.

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Regulated translational bypass of stop
codons in yeast
Tobias von der Haar and Mick F. Tuite
Protein Science Group, Department of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK
Stop codons are used to signal the ribosome to
terminate the decoding of an mRNA template. Recent
studies on translation termination in the yeast Sacchar-
omyces cerevisiae have not only enabled the identifica-
tionofthekeycomponents
machinery, but have also revealed several regulatory
mechanisms that might enable the controlled synthesis
of C-terminally extended polypeptides via stop-codon
readthrough. These include both genetic and epigenetic
mechanisms. Rather than being a translation ‘error’,
stop-codon readthrough can have important effects on
other cellular processes such as mRNA degradation
and, in some cases, can confer a beneficial phenotype
to the cell.
ofthetermination
Translation termination in yeast
Following incorporation of the amino acid encoded by the
last sense codon of an open reading frame, ribosomes end
up with a peptidyl-tRNA attached to the completed protein
product in the P site, and any one of three stop or nonsense
codons (UAA, UAG, UGA) in the A site. Unlike sense
codons, stop codons are not usually decoded by an elonga-
tion factor:tRNA complex but instead are ‘decoded’ by
protein release factors (RFs). In eukaryotes the collective
action of two protein RFs, eRF1 and eRF3, results in
hydrolysis of the bond between peptidyl-tRNA and poly-
peptide and ensuing dissolution of the ribosome:peptidyl-
tRNA:mRNA complex [1].
Much of what has been learnt about the eukaryotic
translation termination mechanism has come from studies
in fungi, especially the yeast Saccharomyces cerevisiae, in
which in vivo studies have been used to complement
in vitro observations made with purified eRFs. Besides
establishing a basic molecular model for translation ter-
mination, these studies have also identified both genetic
and epigenetic mechanisms by which the efficiency of
translation termination can be regulated in S. cerevisiae.
Itwas shownthat reducingtermination efficiencycanhave
positive effects on the ability of cells to respond, and
perhaps adapt longer term, to different environmental
conditions. Regulated termination codon readthrough
could even contribute to the evolution of specific traits in
yeasts and perhaps other fungi. With the increased knowl-
edge of these post-transcriptional mechanisms, it is now
timely to review the emerging literature on how yeast cells
regulate the expression and function of their translation
termination machinery, and to assess what effects such
regulation might have at the molecular and cellular levels.
The molecular mechanism of translation termination
The basic mechanism of translation termination seems to
be similar in all eukaryotic species studied to date. Two
proteins, eRF1 and eRF3 (which in S. cerevisiae are
encoded by the SUP45 and SUP35 genes, respectively),
interact to form the release factor activity in eukaryotes in
vivo, although eRF1 alone is sufficient to achieve polypep-
tide release in vitro. eRF1 is structurally organized into
three domains: N terminal, middle and C terminal [2].
Only the N-terminal and middle domains, in the absence of
eRF3, are necessary to mediate polypeptide chain release
inreconstitutedmodelsystemsinvitro[3].Accordingtothe
currently accepted molecular model (reviewed in ref. [1]),
eRF1 binds directly to a ribosomal A site containing a
stopcodon,where it activatesthe ribosomal peptidyl trans-
ferase centre (PTC) to catalyze the polypeptide release
reaction [4].
Although the N-terminal and middle domains of eRF1
are sufficient for polypeptide release in vitro, the C-term-
inal domains of both eRF1 and eRF3 are required for cell
viability and efficient translation termination in vivo.
eRF3 is a GTP-binding protein in addition to a ribo-
some-dependent and eRF1-dependent
eRF3-mediated GTP hydrolysis and physical binding of
eRF3 to eRF1 are essential parts of the termination reac-
tion [5,6] but the molecular details of the eRF1–eRF3–GTP
interaction are not yet clear as a result of several conflict-
ing datasets [7–10].
GTPase.Both
The role of eRF3-mediated GTP hydrolysis
S.cerevisiaeeRF1wasreportedtobindtoeRF3invitroonly
in the presence of GTP [8], whereas two in vitro studies
conducted with human eRF1 and eRF3 provided evidence
for eRF1 binding to eRF3 in the absence of GTP [7,10]. In
the latter case, formation of the eRF1:eRF3 complex was
found to be a prerequisite for efficient GTP binding
[7,10,11]. Moreover, in the fission yeast Schizosaccharo-
myces pombe, eRF3 binds to GDP in the absence of Mg2+
ions but not in the presence of physiological concentrations
of these ions [9], suggesting that guanidine nucleotide
exchange following GTP hydrolysis occurs spontaneously
in this organism through rapid release of GDP from the
protein. By contrast, human eRF3 binds efficiently to GDP
under physiological Mg2+concentrations [10,11]. These
studies suggested that nucleotide exchange following
GTP hydrolysis could be achieved because, in contrast to
Corresponding author: von der Haar, T. (T.von-der-Haar@kent.ac.uk).
Available online 21 December 2006.
www.sciencedirect.com 0966-842X/$ – see front matter ? 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.12.002

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free eRF3, the eRF1:eRF3complexshows higheraffinityfor
the triphosphate than for the diphosphate, thus effectively
making eRF1 a guanidine nucleotide exchange factor for
eRF3. It is currently not clear whether the differing results
for the fungal and mammalian proteins reflect genuine
species-specific differences in the implementation of trans-
lation termination, or whether they are caused by differ-
ences in the experimental in vitro systems used in these
studies.
Studies using in vitro assays with the human eRFs [12]
suggest a likely model in which eRF3 functions as a proof-
reading factor. Upon binding of an eRF1:eRF3:GTP com-
plex to the ribosomal A site, eRF3 in its GTP-bound form
prevents eRF1 from catalyzing polypeptide release. If the
codon in the A site is a stop codon, conformational rear-
rangements activate the GTPase activity of eRF3. The
resulting eRF3:GDP complex then loses its inhibitory
effect on eRF1 and polypeptide releaseoccurs. The require-
ment for proofreading in translation termination might
stem from the need to distinguish the stop codons UAA,
UGA and UAG from the closely related Trp codon UGG.
Ribosomal complexes with any of these four codons in their
A site are bound relatively efficiently by eRF1 in vitro [13],
although translation termination does not occur at appre-
ciable levels on TGG codons in vivo.
Readthrough of stop-codons
No cognate tRNAs that can decode stop codons exist in
wild-type yeast cells; however, near-cognate tRNAs can
catalyze the incorporation of amino acids in response to
stop codons, albeit with low efficiency [1]. Thus, a ribosome
containing an A site stop codon can undergo one of two
reactions: translation can be terminated by the action of
eRF1–eRF3, or an amino acid can be incorporated into the
nascent peptide bya near-cognate tRNA. In the latter case,
translation elongation will continue to the next in-frame
stop codon or until the ribosome stalls at the 30-end of
the mRNA. The ratio at which these two reactions occur in
the cell is determined by the balance of activities of
the translation termination factors and the translation
elongation machinery.
Examples of regulated stop-codon bypass exist in
several viruses in which the suppression of stop codons is
thought to expand the decoding potential of a limited gen-
ome (e.g. tobacco mosaic virus and Moloney murine leukae-
mia virus [14]), and in Drosophila, in which stop-codon
suppression on the ‘headcase’ (hdc) mRNA contributes to
regulating tracheal development [15,16]. In the case of
yeast, it is now becoming evident that even low levels of
readthrough of natural terminators can have substantial
effects on the function of the encoded protein and on cell
phenotype. In the following section, we review the relevant
mechanisms that enable yeast to regulate global and/or
gene-specific levels of stop-codon readthrough.
Regulation of translation termination efficiency in
S. cerevisiae
Themostdirectwaytomodulatetranslationterminationis
to reduce the cellular levels and/or activity of one or both
eRFs; S. cerevisiae has several mechanisms by which to
achieve this (described here).
Transcriptional regulation of eRF genes
Transcriptome-wide studies show that under many growth
conditions, the ratios of the mRNAs encoding eRF1
(SUP45) and eRF3 (SUP35) are kept relatively constant.
For example, transcription of both genes is repressed
under conditions of heat shock [17,18], long-term exposure
to dithiothreitol [18], nitrogen depletion [18], during the
diauxic shift (the adaptation to the reduction in nutrients
following prolonged logarithmic growth of a culture) [19]
and upon entry into stationary phase [18]. Transcription of
both genes is also induced following release from heat
shock[18].Yet cellscanalsoalter theratioof thesemRNAs
under some other growth conditions; for example, SUP35
but not SUP45 transcription seem to be moderately
induced under conditions of arsenic-induced stress [20],
whereas SUP45 but not SUP35 transcription is repressed
under conditions of short-term (but interestingly, not long-
term) amino acid starvation [18] and oxidative stress [18].
Experiments with different carbon sources also indicate a
trend for SUP35 transcription to increase with growth
rate, whereas no such trend is apparent for SUP45 [18].
Transcriptome analysis has also identified genes that
show similar patterns of transcriptional regulation to
those of SUP35 and/or SUP45 in a range of different
environmental conditions. A high proportion of genes
implicated in ribosomal subunit biogenesis and tRNA
metabolismshowsimilar
SUP35, whereas a high proportion of ribosomal proteins,
translation initiation factors (eIFs) and other factors
related to tRNA metabolism show similar transcription
profiles to SUP45 [18]. Transcriptional regulation of the
SUP45 and SUP35 genes thus seems to be tied to some
extent to the transcriptional regulation of general compo-
nents of the translational machinery.
Direct measurements of the absolute levels of yeast eRF
mRNAs suggest similar numbers of mRNA molecules per
cell [21,22]. By contrast, analyses of the intensity of GFP
fusions of the two proteins in a large-scale study suggest a
roughly sixfold excess of eRF3 over eRF1 [23]. If these
protein abundance data are correct, the divergence
between mRNA and protein ratios for the two factors
indicate a substantial, although as yet unidentified,
post-transcriptional contribution to the regulation of
eRF protein levels in S. cerevisiae.
transcriptionprofiles to
Regulation of release factor activity by post-translational
modifications
The function of many translation initiation and elongation
factors can be modulated by one or more post-translational
modifications (for examples, see Refs [24,25]). It has
recently emerged that eRF1 is also subject to two different
post-translational modifications; methylation and phos-
phorylation. The glutamine residue in the highly con-
served Gly–Gly–Gln (GGQ) motif in domain 2 of eRF1 is
methylated by the MTQ2 gene-encoded methyltransferase
[26,27]. Methylation of the N7atom of glutamine residues
is a rare protein modification and eRF1 is one of only two
proteins known to be subject to this modification in yeast.
The other is the mitochondrial release factor 1 (mRF1),
which is methylated by a mitochondrial-specific methyl-
transferase encoded by the MTQ1 gene [27]. The rarity of
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this modification makes it likely that eRF1 is the sole
substrate for Mtq2 activity, and the severe growth defect
observed upon deletion of the MTQ2 gene presumably
reflects the importance of this modification for eRF1 func-
tion and cell growth. Although it is not known if deletion of
the MTQ2 gene leads to a decrease in termination effi-
ciency, disruption of the MTQ1 gene does lead to an
elevated level of stop-codon readthrough in mitochondria
(i.e. a nonsense suppression phenotype) [27]. The extent to
which cytoplasmic eRF1 is methylated and whether the
modification is dynamic remains to be established.
The regulatory role of phosphorylation of eRF1 is less
clear. S. cerevisiae eRF1 can be phosphorylated at two
serine residues (Ser421and Ser432) by the CK2 protein
kinase [28] and this event is certainly dynamic, showing
an increase under conditions of active cell growth. How-
ever, replacement of Ser421and Ser432with amino acids
that cannot be phosphorylated has minimaleffects on stop-
codon readthrough, suggesting a minimal effect on eRF1
function, at least in the context of translation termination.
To date, no post-translational modifications have been
described for eRF3, although the nature of the N-terminal
residues of this protein indicates a high probability of N-
acetylation [29]. This protein does, however, have one
remarkablepost-translationalproperty,namelytheability
to switch to a self-perpetuating alternative conformational
state known as a prion [30].
Epigenetic regulation of eRF3 activity
The eRF3 of S. cerevisiae seems to be unique in being able
to form large prion-like polymers in certain strains (called
[PSI+])inwhich>90%oftheeRF3moleculesarepresentin
the presumably non-functional prion form (Figure 1). The
presence of the [PSI+] prion can be readily detected by
increased de novo suppression of nonsense mutations in
auxotrophic markers such as ade1-14 and ade2-1 [30], and
the associated termination defect can be detected using in
vitro translation assays [31]. The prion form of eRF3 and
the associated reduced termination efficiency phenotype
are readily transmitted to daughter cells in the absence of
any underlying genetic change in those cells. The remark-
able prion properties of eRF3 therefore provide a novel
epigenetic mechanism for regulating translation termina-
tion efficiency.
Trans-acting factors that impact on eRF activity
eRF1 and eRF3 are the key protein factors responsible for
effecting translation termination and, so far, they are the
only known translation termination-related proteins that
are essential for cell viability. Through a combination of
genetic screens and protein interaction studies in yeast, at
least nine additional gene products have been implicated
in translation termination with either increases or
decreases in their activity that lead to detectable changes
inthefrequencyofstop-codonreadthrough(Table1).Many
of these genes seem to contribute to cellular functions that
are closely related to and potentially controlled by transla-
tion termination. Three of these factors, Upf1p, Upf2p and
Upf3p, are involved in the recognition and rapid degrada-
tion of mRNAs that contain premature stop codons
(reviewed in ref. [32]) while three further factors, Pab1p,
Tpa1p and Pop2p, are involved in regulating the poly(A)
tail lengths and, thus, the stability of translated mRNAs
(see later). Pab1, the poly(A) binding protein, is addition-
ally involved in regulating translation initiation and
mRNA export [33–35]. The multiple connections between
Figure 1. Yeast cells that carry the prion form of eRF3 (Sup35p) show a reduced efficiency of translation termination. (a) When an in-frame stop codon (in this case the UGA
codon) arrives at the ribosomal A site, it is efficiently recognized by the eukaryotic release factor (eRF), which is made up of two subunits: eRF1 (Sup45p) and eRF3 (Sup35p).
This results in release of the polypeptide chain from the peptidyl-tRNA that is positioned at the P site on the ribosome and is associated with the last sense codon (NNN) in
the open reading frame of the mRNA. The synthesis of the wild-type polypeptide will predominate because any near-cognate tRNAs will be outcompeted by the eRF for
binding to the stop codon. Nevertheless, a small fraction of the tRNA-mediated decoding events can occur and this results in the insertion of an amino acid (aa) in response
to the stop codon and leads to the synthesis of the extended polypeptide. (b) In [PSI+] cells, which carry the aggregated prion form of eRF3 (Sup35p), the concentration of
functional eRF3 and hence eRF is greatly reduced. This enables the near-cognate tRNA to more effectively compete for the stop codon with the limited eRF and this leads to
increased synthesis of the extended polypeptide chain. Wild-type cells that do not have the [PSI+] prion are referred to as [psi?] cells.
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factors that affect translation termination and those that
affect the adenylation state of mRNAs suggest that the two
functionsaresomehowinter-related,andthatregulationof
one of them will affect the efficiency of the other.
The remaining three factors identified (Ecm29p/Mtt1p
[36], Itt1p [37] and Ppq1p [38]) have less well-defined roles
in termination. Evidence concerning their specific roles is
largely anecdotal, and no regulatory mechanism involving
any of them has yet been established. Deletion of all three
genes is tolerated with no or only minor reductions in
growth rate, thus suggesting that their roles (if any) in
regulating translation termination are accessory rather
than central, at least under laboratory growth conditions.
Alteration of translation termination by tRNAs and
elongation factors
The low frequency with which an A site stop codon is
decoded by a near-cognate tRNA demonstrates that
eRF-mediated translation termination is much more effi-
cient than near-cognate decoding by the elongation
machinery. However, under conditions in which the elon-
gation factors are more active than normal or in which the
fidelity of codon:anticodon interactions is decreased by
antibiotics or rRNA mutations [1], significant levels of stop
codon readthrough can be observed [39–41]. Accordingly,
overexpression of several genes related to translation
elongation (TEF4, ARC1 and DTD1) increases levels of
stop-codon readthrough in yeast [42], as does the deletion
of ribosome-associated chaperones that are required for
the maintenance of translational fidelity [43]. These
results demonstrate that accuracy in A site decoding,
including the correct decoding of a stop codon as a transla-
tion termination signal, requires a finely tuned balance
between the various translational processes. Any regula-
tory mechanisms that impact on the elongation machinery
can, therefore, potentially modulate the efficiency of trans-
lation termination.
As discussed in the preceding paragraphs, S. cerevisiae
has evolved several potential ways to regulate translation
termination efficiency and the frequency of stop-codon
readthrough. An open question is: how much is the regula-
tion of stop-codon readthrough actually used by cells? If so,
what purpose does it serve in the context of cellular growth
and adaptation? In the following section, we examine the
evidence emerging from studies with yeast concerning
consequences of reduced translation termination efficiency
at both the cellular and molecular levels.
Effects of epigenetic regulation of translation
termination efficiency on yeast
Reducing the efficiency of translation termination in yeast
can have substantial effects on cell phenotype. At its most
extreme, as in the case of certain combinations of muta-
tions that impair termination [44], the cells will die. How-
ever, more subtle, possibly beneficial changes can arise
from smaller reductions in termination efficiency, as found
in [PSI+] strains that contain the prion form of eRF3
(Sup35p).
The [PSI+] prion provides the yeast cell with a novel
epigenetic mechanism through which to modulate transla-
tion termination, but the existence of such a mechanism
raises two important questions: (i) why would such a
mechanism have evolved? (ii) What evolutionary benefit
– if any – does it confer? The first clues to the answers to
these questions came from the observation that [PSI+]
strains show a greater degree of thermotolerance than
their [psi?] counterparts [45]. Subsequently, detailed stu-
dies by True and Lindquist [46,47] showed that there are
several other phenotypes that are modulated by the pre-
senceofthe[PSI+]prion,particularlythosethat mightgive
yeast a selective advantage in certain ecological niches or
when exposed to potentially toxic chemical agents or dif-
ferent carbon and nitrogen sources. For several of the
phenotypes associated with [PSI+], the alterations in cell
phenotype are directly associated with stop-codon read-
through [46].
The [PSI+] prion might, therefore, provide an epigenetic
mechanismthatrevealshiddengeneticvariationwithinthe
yeast genome and provides a ‘one stop’ mechanism through
whichthe organismcanacquire complextraits [46,47]. This
hypothesis has stimulated considerable debate [48–50] and
various counter arguments have been put forward. For
example, the failure to find any ‘wild’ strains of Sacchar-
omyces cerevisiae that contain the [PSI+] prion have been
used to argue that the presence of this prion is actually
deleterious to the yeast cell and hence selected against [51].
It is possible that the absence of [PSI+] prions in natural
strainsofS.cerevisiaesimplyreflectsthefactthatthe[PSI+]
Table 1. Saccharomyces cerevisiae genes implicated in translation termination and stop-codon readthrough
Gene Description of gene producta
SUP45
eRF1, an essential termination factor, decodes the A site stop codon and induces polypeptide release.
SUP35
eRF3, an essential termination factor, eRF1- and ribosome-dependent GTPase, potential proofreading factor that might
control eRF1 activity.
NAM7/UPF1
ATP-dependent RNA helicase, essential for nonsense-mediated decay. Interacts with both eRF3 and eRF1. Deletion
produces an allosuppressor phenotype.
NMD2/UPF2
Involved in nonsense-mediated decay. Interacts with eRF3. Deletion produces an allosuppressor phenotype.
UPF3
Involved in nonsense-mediated decay. Interacts with eRF3. Deletion produces an allosuppressor phenotype.
PAB1
mRNA poly(A) binding protein. Interacts with eRF3. Overexpression produces an antisuppressor effect.
TPA1
Regulates mRNA poly(A) tail length. Interacts with eRF1 and eRF3. Deletion leads to stop-codon suppression.
POP2
Subunit of the deadenylase complex that controls poly(A) tail length and mRNA half life. Deletion leads to stop-codon
suppression.
ECM32/MTT1
ATP-dependent RNA helicase. Interacts with eRF3. Overexpression produces an antisuppressor effect.
PPQ1
Putative PP1 family protein phosphatase. Deletion produces an allosuppressor phenotype.
ITT1
Potential inhibitor of translation termination. Overexpression leads to stop-codon readthrough.
Refs
[76]
[77]
[78]
[78]
[78]
[61]
[63]
[63]
[36]
[38]
[37]
aAntisuppression is a phenotype that shows a reduced efficiency of a defined nonsense suppressor tRNA (i.e. increased efficiency termination). Allosuppression is a
phenotype that shows an increased efficiency of nonsense suppression (i.e. reduced efficiency of termination).
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state only needs to be expressed transiently and one would
expectittobepredominantonlywhenitspresenceconfersa
selective advantage [50].
Although the studies described here have identified
phenotypic changes resulting from increased stop-codon
readthrough, other studies have addressed the molecular
mechanisms that connect alterations in termination effi-
ciency with changes in the gene expression profiles of yeast
cells.
Molecular consequences of reduced termination
efficiency
At a molecular level, the changes in phenotypic adaptation
caused by alterations in termination efficiency are likely to
stem from the collective impact of many different effects.
Translation termination directly or indirectly affects the
production of protein from an mRNA by a variety of
mechanisms (Figure 2) and which of these mechanisms
is relevant for a particular mRNA depends, at least in part,
on sequence features specific to that mRNA.
Production of extended gene products
For any mRNA that contains a second in-frame stop codon
in its 30untranslated region (UTR), readthrough of the
natural terminator and termination at the subsequent in-
framestopsignalproducesapolypeptidewithaC-terminal
extension.
One important restriction on stop-codon readthrough is
the 50and 30sequence context in which a particular in-
frame stop codon is placed. This means that even when
translation termination is impaired, not all termination
codons are decoded with equal efficiency. One major deter-
minant of the ‘leakiness’ of a termination codon is the
nucleotideimmediately 30of thestopsignal inS.cerevisiae;
this can vary the readthrough efficiency on an individual
stop codon by 100-fold [52]. The identity of the six nucleo-
tides upstream and downstream of the stop signal exerts
further, albeit much smaller, effects on termination effi-
ciency [52–54].
A substantial number of yeast genes contain secondary
in-frame stop codons located one, two or three codons
downstream from the first stop [55]. Even though the
protein extensions produced from such mRNAs are short,
they can alter the physical properties of the relevant gene
products by, for example, affecting the thermodynamic
stability or the rates of turnover of the extended protein
(for an example, see Ref. [56]).
In some yeast genes, secondary stop codons are located
further downstream of the natural terminator [57], and in
these cases considerable functional alterations can be
conferred on the extended gene product. This is best illu-
strated by the yeast PDE2 gene, in which a C-terminal
extension of 21 amino acids (under conditions of decreased
termination efficiency) leads to destabilization of the pro-
tein [58]. PDE2 translation is naturally terminated in a
leaky (i.e. weak) nucleotide context, and the wild-type gene
product is expressed at relatively low steady-state levels.
Insertion of a strong (non-leaky) nucleotide context for the
natural terminator leads to a substantial increase in
steady-state levels of the wild-type protein. Because the
PDE2 gene product is a cAMP phosphodiesterase, a strong
stop codon context correlates with decreased levels of
intracellularcAMPandconfersincreasedstresssensitivity
to the cells. PDE2 is thus an example of an individual gene
product in S. cerevisiae where alterations in stop-codon
readthrough are experimentally linked to phenotypic var-
iations. Whether this is more widely used in yeast and
other eukaryotes remains to be established, although the
existence of pairs of open reading frames in the yeast
genome that are separated by an in-frame stop codon
[59] would indicate that there are likely to be more exam-
ples.
Alterations of mRNA levels through regulation of mRNA
turnover
In yeast, the stability of mRNAs is dependent on the rate
andextentofpoly(A) tail shortening(reviewedinRef.[60]).
Most yeast mRNAs start their lives with long poly(A) tails
and are associated with multiple copies of the poly(A)
binding protein, Pab1p. Active translation leads to pro-
gressive poly(A) shortening through the action of a nucle-
ase complex and once tails are shortened to a length of ?10
Figure 2. Effects of stop-codon readthrough on gene expression and cellular
function. Readthrough of premature stop codons (black TER) will result in
restorationofsynthesis ofthe full-length
suppression) and will also prevent detection and degradation of this mRNA by
the nonsense-mediated decay (NMD) factors. Readthrough of a normal ORF stop
codon (red TER) results in expression of a C-terminally extended polypeptide only
if a second in-frame stop codon is present on that mRNA. Because normal
translation termination interfaces with deadenylation of the mRNA, such a
readthrough event also slows down shortening of the poly(A) tail and thus
reduces rates of mRNA turnover. If there is no further stop codon present on the
mRNA [i.e. if readthrough involves the last in-frame stop codon of an mRNA (blue
TER)], the ribosome will continue to elongate to the end of the message and stall
there. Such complexes are detected and the corresponding mRNA is degraded by
the non-stop decay (NSD) factors. Where translation termination becomes less
efficient and ribosomes pause on stop codons, the pausing ribosome might also
recruit endonuclease activities that lead to immediate inactivation of the message:
no-go decay (NGD). The combination of these effects will lead to changes in the
transcriptome and proteome, and might ultimately lead to changes in cellular
function and phenotype. If stop-codon readthrough is caused by alterations in
eRF1 or eRF3 activity, changes in cellular function can also be directly caused
through non-translational roles of the termination factors.
gene product (i.e. nonsense
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adenylic acid residues, this triggers remodelling of protein
complexes bound to the mRNA. This, in turn, renders the
mRNA a target for degradation.
Recent results suggest that translation termination
could exert direct control over the rate of mRNA dead-
enylation.eRF3(Sup35p)bindstoPab1p[61,62],andyeast
strains in which this interaction is impaired show reduced
rates of mRNA deadenylation and increased mRNA stabi-
lity [45]. Moreover, deadenylation seems to be dependent
on normal translation termination because mutations in
eRF3 that decrease termination efficiency (but show no
reduced binding to Pab1p) also stabilize mRNAs [8]. The
fact that eRF3 binding to Pab1p prevents the oligomeriza-
tion of Pab1p on poly(A) tails [62] suggests a possible
molecular mechanism by which contacts between these
two proteins during a translation termination event
release Pab1p from the poly(A) tail, thus providing access
for the deadenylation factors. eRF3 also interacts with a
second deadenylation factor, Tpa1p, and defects in the
deadenylase complex produce a stop-codon readthrough
phenotype [63]. A tight connection therefore exists
between translation termination and deadenylation and
mRNA turnover.
Alterations of mRNA levels through surveillance
pathways
Several surveillance pathways exist in eukaryotic cells to
identify aberrant mRNAs and rapidly destroy them.
mRNAsthatcontainaprematurestopcodonarerecognized
by the nonsense-mediated decay (NMD) machinery, which
involves the canonical translation termination factors in
conjunction with dedicated NMD factors (see earlier;
reviewedinref.[32]).AsecondpathwayoperatesonmRNAs
that contain no in-frame stop codon, on which ribosomes
translate through to the 30-end of the mRNA and stall. The
resulting dead-end complexes are recognized and rapidly
degradedbythenon-stopdecay(NSD)pathway(reviewedin
Ref. [64]). mRNAs on which a ribosome has stalled during
Figure 3. The putative interacting partners for eRF1 and eRF3 cluster into functional categories. Physical interactions for yeast eRF1 and eRF3 reported in the literature were
retrieved from the BIOGrid database [57]. Circles represent the interaction partners, and are colour-coded according to functional category (see colour key in figure).
Connecting lines between partners represent the interaction: interactions that have been reported in several studies and corroborated by different experimental techniques
are in red; large-scale interaction studies that have not yet been confirmed by independent experimental approaches (reported in one study and by one technique only) are
in black. Interactions are shown in detail where several functionally related proteins are reported to interact with the eRFs; other reported interacting partners are also listed.
A large number of interactions with functionally related proteins indicate that the eRFs might have important non-translational roles in the respective process, or that the
process in question might exert control over the efficiency of translation termination.
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elongationaresubjecttoendonucleolyticcleavageandrapid
degradation through a third pathway, the no-go decay
(NGD) pathway [65].
Alterations in termination efficiency decrease the
efficiency of surveillance by NMD because they can mask
the presence of stop codons, albeit transiently, from the
NMD factors. Surprisingly, even low levels of readthrough
(?0.5%) can significantly reduce NMD-mediated turnover
of an mRNA [66], although other data indicate that the
stabilized mRNAs will be degraded by the NMD pathway
once a premature termination event actually occurs
[67,68]. Readthrough of the last stop-codon of an mRNA
will lead to ribosome stalling at the 30-end and activation of
the NSD pathway, resulting in accelerated decay. Such
modulation of mRNA levels can make a considerable con-
tribution to some of the phenotypic variation associated
with altered stop-codon readthrough in the [PSI+] and
[psi?] states, because the deletion of factors essential for
the NSD pathway modulates several of the [PSI+] related
phenotypes [69].
Reducing the efficiency of translation termination also
induces the NGD surveillance pathway. Reduced eRF
activity allows for the increased decoding of stop codons
by near-cognate tRNAs, but decoding through near-cognate
tRNAs is less efficient than translation elongation through
cognatetRNAs.Ina cellinwhichtranslationterminationis
impaired,ribosomeswill,therefore,temporarilystallonthe
stop codons and this can be sufficient to induce endonucleo-
lytic cleavage and subsequent degradation of the bound
mRNA [65]. Reducing activity of one or other of the eRFs
would thus lower the steady-state levels of all mRNAs that
are subject to this form of surveillance.
In summary, the regulation of mRNA via stop-codon
readthrough levels is the result of overlapping and partly
opposing effects on deadenylation in addition to the var-
ious surveillance pathways.
Phenotypic effects arising from regulation of non-
translational functions of eRFs
Mutations in yeast eRFs can cause pleiotropic effects on a
cell including cytoskeletal defects [70], abnormal chromo-
some segregation [71], cell cycle defects [72] and respira-
tory deficiency [73]. It remains unclear as to whether these
effects are the direct result of impairment in a translation-
unrelated function of the eRFs, or as a result of indirect
effects that arise from the synthesis of extended polypep-
tide chains because of stop codon readthrough. The only
non-translational process for which direct involvement of
an eRF has been conclusively shown is cytokinesis, in
which an interaction between eRF1 and the myosin light
chain Mlc1p are essential for proper functioning [70].
Whether such contacts occur as part of the translation
termination process or involve a sub-population of eRF1
molecules that is not involved in translation termination
remains to be established. Nevertheless, the existing data
indicate that regulation of eRF activity can potentially
affect cytoskeletal functions independently of stop-codon
readthrough [58].
Genome-wideinteractionstudies haveuncovered a large
numberoffurtherinteractionsforbotheRF1andeRF3inS.
cerevisiae (summarized in the BIOGrid database [74]).
Although many of these reported interactions come only
from large-scale studies and are otherwise unconfirmed, it
is interesting to note that the putative interacting proteins
cluster into functional categories. In particular, proteins
related to cytoskeletal functions, to the generation of
tRNAs and rRNAs, and to vacuolar function are prevalent
(Figure 3). The abundance of these putative interacting
partners suggests that there might be further, hitherto
undiscovered non-translational functions of the eRFs.
Concluding remarks and future perspectives
Several molecular mechanisms have emerged from recent
studies that could cause a remodelling of specific or global
cellular activities under conditions of reduced termination
efficiency.Althougheffectsonadaptationthatmightbethe
consequence of such mechanisms have been demonstrated
[45–47], it is still unclear whether or how the regulation of
termination efficiency is actually used for controlling cel-
lular growth and adaptation in yeast. For example, the
efficiency of translation termination seems to vary little
during the transition from logarithmic growth to station-
ary phase [75]. However, there is a shortage of studies
addressing the question of whether termination efficiency
is actually regulated under different environmental con-
ditions, and therefore further work is needed (Box 1).
Another area in which information is lacking concerns
the balance of the various downstream effects caused by
stop-codon readthrough. The most important develop-
ments have been the identification of cellular pathways,
such as mRNA turnover and surveillance that are affected
by translation termination. The convoluted and partly
opposing effects of stop-codon readthrough on mRNA
half-lives make it currently impossible to predict the mag-
nitude of changes in mRNA half-life for an individual
transcript. It is likely that this problem will only be solved
once quantitative kinetic models become available for the
processes of both translation and mRNA decay.
Acknowledgements
T.v.d.H. is supported by a Wellcome Trust Research Career Development
Fellowship (075438/Z/04/Z). Research in the M.F.T. laboratory is
supported by the Biotechnology and Biological Sciences Research
Council, the Wellcome Trust and the European Commission (via
contract LSHM-CT-2003-503330).
Box 1. Open questions
? Ribosome recycling. How does the ribosome-recycling step
(i.e. the release of the ribosome from the mRNA) function in
eukaryotes? Although this step is well-characterized in prokar-
yotes, where it requires a dedicated ribosome recycling factor [79]
(RRF), there is currently no molecular model available to explain
the dissolution of the post-polypeptide release of ribosome:-
tRNA:mRNA complexes during translation termination in eukar-
yotes.
? Regulation. Is translation termination efficiency constant through-
out all growth conditions, or is it regulated and – if so – does its
regulation contribute to cellular growth and adaptation?
? Non-translational roles. What is the extent and nature of the
non-translational roles of the eRFs? Are such roles intricately
linked to translation termination, or do they involve cellular pools
of the factors that are separate from eRFs that participate in
termination?
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phases inSaccharomyces
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