point-of-view
RNA Biology 7:5, 548-550; September/October 2010; © 2010 Landes Bioscience
Translation termination
New factors and insights
Claudia Baierlein and Heike Krebber*
Georg-August-Universität Göttingen; Institut für Mikrobiologie und Genetik; Göttingen, Germany
I
Key words: translation, termination,
eRF1, eRF3, Dbp5, Rli1, Gle1
Submitted: 04/14/10
Revised: 06/08/10
Accepted: 06/10/10
Previously published online:
www.landesbioscience.com/journals/
rnabiology/article/12686
*Correspondence to: Heike Krebber;
Email: [email protected]
548
n eukaryotes, translation termination
requires two eukaryotic release factors, eRF1 and eRF3. eRF1 is required
for recognition of the stop codon and
eRF3 supports the polypeptide chain
release in a GTP dependent manner.
Recently, several new players in translation termination have been identified.
The DEAD-box RNA helicase Dbp5
has been shown to support eRF1 in stop
codon recognition, possibly by proper
placement of the release factor on the
termination codon. Upon its dissociation
from eRF1, Dbp5 allows the entry of the
second termination factor eRF3 into the
complex. Further, the Dbp5 interacting protein Gle1 and its co-factor inositol hexakisphosphate (IP6) have been
shown to participate in the termination
process. Dbp5 and Gle1 are well known
for their function in mRNA export from
the nucleus to the cytoplasm. Most interestingly, also the ATP binding cassette
(ABC) protein Rli1, which requires the
mitochondrial and cytosolic Fe/S protein
biogenesis machineries for its assembly,
has recently been shown to function in
translation termination and recycling
of the ribosomes. Rli1 physically and
genetically interacts with both, eRF1 and
eRF3. Together, all of these novel termination factors modulate in association
with the release factors more accurate
stop codon recognition and these findings broaden our knowledge about the
final steps in translation.
Protein synthesis is a well conserved process in every kingdom of life. mRNA
translation is highly regulated and cells
can attune their proteome to specific
conditions more rapidly by tuning translation than through modulation of an earlier
step in gene expression like transcription.
Therefore, in different steps of translation proteins are recruited or dissociate
from complexes to optimize the process.
Translation can be separated into initiation, elongation, termination and ribosome recycling.1 Translation initiation is
highly regulated and requires many different proteins.2,3 The formation of the multi
protein complex at a start codon of an
mRNA does not only require the monosome but includes also several different
factors that control the process and finally
trigger translation elongation to built the
polypeptide chain.2 Translation termination finally occurs when the ribosome has
reached a stop codon. Two classes of release
factors (RFs) mediate this process: Class I
RFs recognize and bind to the stop codons
and class II RFs are GTPases that enhance
the activity of class I RF.4 Whereas in bacteria different stop codons are recognized
by different termination factors (RF1 for
UAA/UAG and RF2 for UAA/UGA),
eukaryotes require only one protein for
the recognition of all three stop codons,
the eukaryotic release factor 1 (eRF1)
encoded by SUP45 in Saccharomyces cerevisiae.5,6 eRF1 is a functional mimicry of
the tRNAs and through its binding to
the A site of the ribosome it triggers the
hydrolysis of the peptidyl-tRNA, resulting
in the peptide chain release.7 The second
eukaryotic release factor eRF3, encoded
by SUP35 in yeast, accelerates this process in a GTP-dependent manner.8 Finally
ribosome recycling occurs. This process is
mediated by components of the initiation
complex, namely, eIF3, eIF1 and eIF1A.9
RNA BiologyVolume 7 Issue 5
point-of-View
point-of-view
Figure 1. Model for translation termination in eukaryotes. When a translating ribosome has reached a stop codon, it arrests at this position. The tRNA
mimicking eukaryotic release factor 1 (eRF1) recognizes the stop codon and its binding is supported by the DEAD-box RNA helicase Dbp5, possibly
by rearranging the surrounding RNA/protein structures to fit eRF1 into its correct position. For this conversion the ATPase activity of Dbp5 is required,
which is stimulated by the cofactors Gle1 and inositol hexakisphosphate (IP6). After this remodeling process Dbp5 dissociates, which in turn allows the
entrance of GTP-bound eRF3 into the ribosome complex. It is currently unclear if Gle1 dissociates or remains bound to the ribosome. The entry point
of Rli1/ABCE1 is currently unknown, but it is likely that it supports the termination accuracy in pre-termination complexes (termination complexes
before polypeptide chain release). eRF3, which is bound to eRF1 subsequently hydrolyzes GTP, resulting in the polypeptide chain release. For recycling
of the ribosomal subunits, eIF3 associates with its stimulating factors eIF1 and eIF1A and dissociate supported by NTP hydrolysis through Rli1/ABCE1,
the RNA-bound ribosome into free 60S, 40S, tRNA and mRNA. If the initiation factors and Rli1 and/or Gle1 remain bound to ribosomal subunits for
initiation is unclear and remains to be shown.
While eIF3 acts as the major factor in dissociating post-termination complexes into
60S and t- and mRNA-bound 40S, eIF1
promotes tRNA release and eIF3j mediates the mRNA dissociation.9
It is apparent that translation initiation is highly regulated and many proteins
have been identified that participate and
regulate this process. In contrast to that,
the regulation of translation termination
seems much simpler. Therefore, the recent
discovery of several novel termination
factors was surprising (Fig. 1). The first
novel termination factor that was identified in the last years was a DEAD-box
RNA helicase termed Dbp5/Rat8.10 Dbp5
and its ATPase stimulating cofactors Gle1
and inositol hexakisphosphate (IP6), have
established roles in mRNA export from the
nucleus to the cytoplasm.11-13 Interestingly,
Dbp5 is also essential for translation termination as mutants are defective in
stop codon recognition and show genetic
interactions with both termination factors
eRF1 and eRF3.10 Strikingly, the helicase
physically interacts with eRF1, but not
with eRF3 and it was shown that mutants
of Dbp5 are defective in the recruitment of
eRF3 into termination complexes. These
findings led to a model in which Dbp5 and
eRF1 interact to promote efficient translation termination. The helicase activity of
Dbp5 might remodel the mRNA/protein
complex to allow proper eRF1 positioning on the stop codon and thus an efficient termination reaction. Subsequent
www.landesbioscience.com
dissociation from eRF1 is followed by the
entry of eRF3 into the complex, which
promotes polypeptide chain release (Fig.
1).10 Remarkably, Bolger et al. found that
Gle1, the cofactor of Dbp5, also functions
in an IP6 -dependent manner in translation termination.14 It is part of the termination complex, it interacts preferentially
with eRF1 and mutants of GLE1 show
increased read-through activity as well
as genetic interactions with mutants of
eRF3 and the eIF3 translation initiation
factor mutant nip1-1. Interestingly, gle1
mutants have defects in initiation, whereas
strains lacking IP6 do not, suggesting that
Gle1 functions together with IP6 and the
DEAD-box protein Dbp5 in the regulation of translation termination, but independent of IP6 in translation initiation.14
Rli1 (ABCE1 in mammalia) is another
novel tanslation termination factor discovered recently.15 Rli1 is an essential
iron-sulfur (Fe/S) containing protein
that was identified as an RNase L inhibitor. It belongs to the ATP-binding cassette (ABC) proteins and requires the
mitochondrial and cytosolic Fe/S protein biogenesis machineries for its assembly.16,17 A function of Rli1 in translation
initiation was suggested earlier, because
it has been shown that Rli1 associates
with polyribosomes, with the initiation
factors eIF2, eIF5 and different subunits
of eIF3. Additionally Rli1 is required for
the formation and stabilization of the 43S
pre-initiation complexes.18,19 Interestingly,
RNA Biology
a very stable interaction was detected
between Rli1 and eIF3, especially with
the subunit eIF3j (Hcr1 in S. cerevisiae),19
which is also involved in ribosome recycling.9 Additionally Hcr1 stimulates the
assembly of the eIF3 complex and its
stable association with the 40S ribosomal
subunit supports the formation of the 43S
pre-initiation complex.20
The connection of Rli1 with translation
termination came from a yeast two-hybrid screen in which one of its interacting proteins turned out to be eRF1.15 In
vivo and in vitro studies further revealed
that Rli1 physically interacts with both,
eRF1 and eRF3. Interestingly, the interaction domain of Rli1 for its interaction
with eRF1 and Hcr1 was localized to its
second ABC domain. Moreover, genetic
interactions were uncovered between a
strain depleted for Rli1 and mutant eRF1
(sup45-2) or eRF3 (sup35-21). While the
combination of low Rli1 with sup45-2 is
synthetically lethal, its combination with
sup35-2 is indeed impaired, but clearly
viable, suggesting that Rli1 might regulate
the termination process through eRF1.
Importantly, downregulation of RLI1
leads to defects in stop codon recognition, also seen in mutants of eRF1, eRF3
and Dbp5. Overexpression of RLI1 on the
other hand suppresses the readthrough
defects of sup45-2. It was further shown
that although the Fe/S cluster is not
required for the interaction of Rli1 with
eRF1 or Hcr1, it is required for its activity
549
in translation termination, as an Fe/S cluster mutant of RLI1 does not suppress the
readthrough defects of the eRF1 mutant
sup45-2.15 These data clearly suggest that
Rli1 modulates translation termination in
yeast.
Strikingly, in a concurrent study the
homolog of Rli1 in higher eukaryotes,
ABCE1 was shown to strongly enhance
ribosome recycling.21 While ribosome
recycling is accomplished to some extent
by eIF3, eIF1 and eIF1A,9 ABCE1
enhances subunit dissociation in various conditions.21 Its ribosome association
depends on eRF1 and the ribosome dissociation requires NTP-hydrolysis by
ABCE1. Interestingly, ABCE1 was also
found on pre-termination complexes,
which might suggest also earlier functions
of the mammalian protein in translation
termination. Thus, these novel functions
of Rli1/ABCE1 in translation termination and ribosome recycling have opened
a new and deeper look into these late steps
of translation. It is tempting to speculate
that Rli1/ABCE1 might also provide a
functional regulatory link between termination, recycling and initiation, since it
interacts with eRF1 and Hcr1/eIF3j.
Functional homologues of the three
novel termination factors in prokaryotes have not been identified so far.
Nevertheless, significant structural homologues exist for Rli1 in archaebacteria with
overall amino acid identities of up to 49%.
In contrast, no homology of Gle1 to bacterial proteins is apparent. As helicases in
general have certain sequence similarities,
homology searches with the RNA helicase
Dbp5 retrieved several bacterial helicases,
which exhibit identities of up to 38%.
Thus, it is unclear if bacterial translation
termination requires more than the three
release factors. It is very well conceivable
550
that the three newly identified termination
factors are only required for the sophisticated 80S ribosome and an advanced and
more complicated process in eukaryotic
cells.
Taken together the previous very
limited number of translation termination factors now significantly increased
in eukaryotes by the identification of
Rli1/ABCE1, Dbp5 and Gle1-IP6 (Fig.
1). Unraveling their functions allowed
a deeper look into the mechanisms of
translation termination and its regulation.
Termination is not an autarkic process
mediated simply by basal release factors 1
and 3, but rather complex and interwoven
with recycling and translation initiation.
Acknowledgements
This work was funded by grants and a
Heisenberg fellowship of the Deutsche
Forschungsgemeinschaft awarded to H.K.
References
1. Kapp LD, Lorsch JR. The molecular mechanics of
eukaryotic translation. Annu Rev Biochem 2004;
73:657-704.
2. Jackson RJ, Hellen CU, Pestova TV. The mechanism
of eukaryotic translation initiation and principles of
its regulation. Nature reviews 2010; 11:113-27.
3. Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: mechanisms and
biological targets. Cell 2009; 136:731-45.
4. Jacobson A. The end justifies the means. Nature
structural & molecular biology 2005; 12:474-5.
5. Frolova L, Le Goff X, Rasmussen HH, Cheperegin
S, Drugeon G, Kress M, et al. A highly conserved
eukaryotic protein family possessing properties
of polypeptide chain release factor. Nature 1994;
372:701-3.
6. Scolnick E, Tompkins R, Caskey T, Nirenberg M.
Release factors differing in specificity for terminator codons. Proceedings of the National Academy
of Sciences of the United States of America 1968;
61:768-74.
7. Polacek N, Gomez MJ, Ito K, Xiong L, Nakamura Y,
Mankin A. The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of
the nascent peptide during translation termination.
Molecular cell 2003; 11:103-12.
8. Alkalaeva EZ, Pisarev AV, Frolova LY, Kisselev LL,
Pestova TV. In vitro reconstitution of eukaryotic
translation reveals cooperativity between release factors eRF1 and eRF3. Cell 2006; 125:1125-36.
9. Pisarev AV, Hellen CU, Pestova TV. Recycling of
eukaryotic posttermination ribosomal complexes.
Cell 2007; 131:286-99.
10.Gross T, Siepmann A, Sturm D, Windgassen M,
Scarcelli JJ, Seedorf M, et al. The DEAD-box RNA
helicase Dbp5 functions in translation termination.
Science New York, NY 2007; 315:646-9.
11. Cole CN, Scarcelli JJ. Transport of messenger RNA
from the nucleus to the cytoplasm. Curr Opin Cell
Biol 2006; 18:299-306.
12.Lund MK, Guthrie C. The DEAD-box protein
Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim. Molecular cell 2005;
20:645-51.
13.Weirich CS, Erzberger JP, Flick JS, Berger JM,
Thorner J, Weis K. Activation of the DExD/H-box
protein Dbp5 by the nuclear-pore protein Gle1 and
its coactivator InsP6 is required for mRNA export.
Nature cell biology 2006; 8:668-76.
14. Bolger TA, Folkmann AW, Tran EJ, Wente SR. The
mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation. Cell
2008; 134:624-33.
15. Khoshnevis S, Gross T, Rotte C, Baierlein C, Ficner
R, Krebber H. The iron-sulphur protein RNase
L inhibitor functions in translation termination.
EMBO reports 11:214-9.
16.Kispal G, Sipos K, Lange H, Fekete Z, Bedekovics
T, Janaky T, et al. Biogenesis of cytosolic ribosomes
requires the essential iron-sulphur protein Rli1p and
mitochondria. The EMBO journal 2005; 24:589-98.
17. Lill R. Function and biogenesis of iron-sulphur proteins. Nature 2009; 460:831-8.
18.Dong J, Lai R, Nielsen K, Fekete CA, Qiu H,
Hinnebusch AG. The essential ATP-binding cassette
protein RLI1 functions in translation by promoting
preinitiation complex assembly. The Journal of biological chemistry 2004; 279:42157-68.
19. Yarunin A, Panse VG, Petfalski E, Dez C, Tollervey
D, Hurt EC. Functional link between ribosome
formation and biogenesis of iron-sulfur proteins. The
EMBO journal 2005; 24:580-8.
20.Valasek L, Phan L, Schoenfeld LW, Valaskova V,
Hinnebusch AG. Related eIF3 subunits TIF32 and
HCR1 interact with an RNA recognition motif in
PRT1 required for eIF3 integrity and ribosome binding. The EMBO journal 2001; 20:891-904.
21. Pisarev AV, Skabkin MA, Pisareva VP, Skabkina OV,
Rakotondrafara AM, Hentze MW, et al. The role
of ABCE1 in eukaryotic posttermination ribosomal
recycling. Molecular cell 2010; 37:196-210.
RNA BiologyVolume 7 Issue 5