lessons not learned fromtranslation

7/28/2019 Lessons Not Learned Fromtranslation

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Lessons (not) learned from mistakes about translation

Marilyn Kozak

Department of Biochemistry, Robert Wood Johns on Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

Received 9 July 2007; received in revised form 16 August 2007; accepted 27 August 2007

Received by A.J. van Wijnen

Available online 5 September 2007

Abstract

Some popular ideas about translational regulation in eukaryotes have been recognized recently as mistakes. One example is the rejection of a

long-standing idea about involvement of S6 kinase in translation of ribosomal proteins. Unfortunately, new proposals about how S6 kinase might

regulate translation are based on evidence that is no better than the old. Recent findings have also forced rejection of some popular ideas about the

function of sequences at the 3 end of viral mRNAs and rejection of some ideas about internal ribosome entry sequences (IRESs). One long-held

belief was that tissue-specific translation via an IRES underlies the neurotropism of poliovirus and the attenuation of Sabin vaccine strains. Older

experiments that appeared to support this belief and recent experiments that refute it are discussed. The hypothesis that dyskeratosis congenita is

caused by a defect in IRES-mediated translation is probably another mistaken idea. The supporting evidence, such as it is, comes from a mouse

model of the disease and is contradicted by studies carried out with cells from affected patients. The growing use of IRESs as tools to study other

questions about translation is discussed and lamented. The inefficient function of IRESs (if they are IRESs) promotes misunderstandings. I explain

again why it is not valid to invoke a special mechanism of initiation based on the finding that edeine (at very low concentrations) does not inhibit

the translation of a putative IRES from cricket paralysis virus. I explain why new assays, devised to rule out splicing in tests with dicistronic

vectors, are not valid and why experiments with IRESs are not a good way to investigate the mechanism whereby microRNAs inhibit translation.

2007 Elsevier B.V. All rights reserved.

Keywords: Translational control; S6 kinase; IRES; Edeine, Poliovirus translation; 14-3-3; dyskeratosis congenita; microRNA; 3 UTR

Science, my lad, is made up of mistakes, but they are

mistakes which it is useful to make because they lead little

by little to the truth.

Jules Verne

1. Introduction

It is not useful, however, to keep making the same mistakes. Toprevent repetition, it helps to know how an erroneous idea initially

took hold and how it was eventually recognized as wrong. With

that as the goal, this review discusses some once-popular ideas

about translational regulation that recently were found to be mis-

taken. The flaws that led to these misunderstandings still pervade

the literature, suggesting the lessons have not yet taken hold.

The first topic (Section 2) concerns the regulatory effects of a

well-known kinase (S6K) thattargets ribosomal protein S6 (rpS6).An old hypothesis linking translation of ribosomal proteins to

phosphorylation of rpS6 persisted for decades, although it was

based only on correlative evidence. Recent studies undertaken to

identify new targets for S6K again make the mistake of relying

more on correlations than on meaningful functional tests.

Section 3 is about unusual mechanisms of translation thought

to be mediated by sequences near the 3 end of viralRNAs. Recent

evidence disproves a model for initiation of translation involving

the tRNA-like sequence at the 3 end of turnip yellow mosaic virus

(TYMV) RNA. An idea about regulation of translation via a

protein that binds the 3 end of rotavirus mRNAs has also been

Gene 403 (2007) 194203

www.elsevier.com/locate/gene

Abbreviations: CrPV, cricket paralysis virus; DC, dyskeratosis congenita;

eIF, eukaryotic initiation factor; Fluc, firefly luciferase; HCV, hepatitis C virus;

HIV, human immunodeficiency virus; IRES, internal ribosome entry sequence;

mTOR, (mammalian) target of rapamycin; NSP3, nonstructural protein 3; PABP,

poly(A) binding protein; PDCD4, programmed cell death protein 4; PTB,

polypyrimidine tract binding protein; Rluc, Renilla luciferase; rpS6, ribosomal

protein S6; S6K, rpS6 kinase; TOP, 5-terminal oligo-pyrimidine tract; TYMV,

turnip yellow mosaic virus; XIAP, X-linked inhibitor of apoptosis. Tel.: +1 732 235 5355; fax: +1 732 235 5356.

E-mail address: [email protected].

0378-1119/$ - see front matter 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2007.08.017
mailto:[email protected]://dx.doi.org/10.1016/j.gene.2007.08.017http://dx.doi.org/10.1016/j.gene.2007.08.017mailto:[email protected]


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retested and rejected. Even before these corrections came out last

year, deficiencies in the original experiments were obvious

(Kozak, 2004), albeit not obvious enough to keep the erroneous

ideas from entering some textbooks (Flint et al., 2004).

Section 4 concerns mRNA sequences, called IRES elements,

that purportedly mediate internal entry of ribosomes. Advocates

of the internal initiation hypothesis are not yet willing to acknowl-edge many of the experimental flaws pointed out by others

(Kozak, 2003, 2005), but advocates do admit that some long-held

ideas about poliovirus translation are incorrect. Recent experi-

ments revealed, for example, that poliovirus is not translated

better in neuronal cells than in other tissues, and Sabin vaccine

strains are not attenuated specifically in neuronal cells. Other

misunderstandings involve IRESs that purportedly exist in

cellular mRNAs. This idea was invoked to explain the

pathological effects of mutations in the dyskerin (DKC1) gene,

but closer study of cells from affected patients suggests an

alternative mechanism unrelated to IRESs. A postulated con-

nection between IRES-mediated translation and a protein called14-3-3, which is frequently lost in tumors, is another case where

the evidence falls far short of what is claimed.

The growing use of putative IRESs as tools to test other ideas

about translation (Section 5) makes it important to think clearly

about this subject. The discussion herein of admitted and

possible misunderstandings might help.

2. How does S6K regulate translation (if it does)?

2.1. An old hypothesis about translation of mRNAs that encode

ribosomal proteins is wrong

It was long believed that, in response to mitogens, phosphor-ylation of rpS6 by the eponymous kinase stimulates translation of

mRNAs thatbeara 5-terminal oligo-pyrimidine tract (TOP). This

hypothesis is no longer tenable, inasmuch as translation of TOP

mRNAs was perfectly normal in knock-in mice whose rpS6 gene

contains serine-to-alanine substitutions at all five sites normally

phosphorylated by S6K (Ruvinsky et al., 2005). The overall rate

of translation in liver cells from these mice was actually higher

than normal (2.5-fold). In a complementary experiment, S6K-

knockoutmice weretested; and again, translation of TOPmRNAs

was not impaired (Pende et al., 2004).

While nearly everyone now admits that the hypothesis linking

S6K and rpS6 to translation of TOP mRNAs was wrong, no onetalks about how the misunderstanding became entrenched. The

answer is that the evidence never went beyond showing a

correlation: when resting cells were stimulated to resume

growing, mRNAs were recruited onto polysomes and rpS6

became phosphorylated (Gressnerand Wool, 1974;Thomas et al.,

1980); and the recruitment onto polysomes depended on 5 TOP

sequences (Levy et al., 1991; Schwab et al., 1999). No one

attempted to define the mechanism. Small differences in activity

between phosphorylated and nonphosphorylated ribosomes were

occasionally reported (Duncan and McConkey, 1982), but

functional effects of phosphorylation were not seen consistently

(Mastropaolo and Henshaw, 1981). People simply found new

ways to demonstrate a correlation between S6K and translation of

TOP mRNAs (Jefferies et al., 1997; Terada et al., 1994). The

complexity of mammalian signaling pathways makes it hard to

interpret those findings, however. The rapamycin-sensitive kinase

(mTOR) that activates S6K induces elevated production of all

components of the translational machinery (e.g. transcription and

processing of rRNAs increases; Hannan et al., 2003) and many

other growth-related processes are stimulated (Wullschleger et al.,2006). The bottom line is that, although mTOR somehow brings

aboutan increasein translation of TOP (and many other) mRNAs,

S6K and phosphorylation of rpS6 are not involved.

2.2. New ideas about how S6 kinase regulates translation are

based on flimsy evidence

The new ideas focus on translation factors. Holz et al. (2005)

postulate that phosphorylation of eukaryotic initiation factor

(eIF)4B by S6K is regulated by binding of S6K to eIF3. The

evidence boils down to showing that S6K associates and

dissociates from eIF3 in a way that correlates with activation ofS6K by mTOR. No hard evidence supports the hypothesis that

release of activated S6K from eIF3 puts the kinase in position

to act on rpS6 and eIF4B, and some evidence contradicts that

idea: the small fraction of eIF3 found to be associated with S6K

was not associated with ribosomes.

The functional consequence of eIF4B phosphorylation is also

dubious. Phosphorylation was shown to increase the amount of

eIF4B that co-immunoprecipitates with eIF3 (Holz et al., 2005,

Fig. 4B; Shahbazian et al., 2006, Fig. 6), but that is not a test of

function. Raught et al. (2004) say a mutation in eIF4B that

prevents phosphorylation by S6K abolishes the activity of the

factor, but the activity they monitored was remarkably artificial:

over-expression ofwild typeeIF4B was found to inhibittranslationof a co-transfected reporter gene, while over-expression of

nonphosphorylatable eIF4B did not inhibit translation.

The other newly identified substrate for S6K is programmed

cell death protein 4 (PDCD4), which has been described as an

inhibitor of eIF4A. There is compelling evidence that phos-

phorylation by S6K promotes degradation of PDCD4 (Dorrello

et al., 2006) but no convincing evidence that PDCD4 regulates

translation. PDCD4 does bind to eIF4A, but over-expression of

PDCD4 caused only a two-fold reduction in translation of a

reporter gene (Yang et al., 2003). These marginal findings

demonstrated by using a synthetic mRNA and over-expressing

PDCD4 do not justify the assertion that PDCD4 is a noveltumor suppressor that inhibitstranslation of proteins that are

essential for neoplastic transformation (LaRonde-LeBlanc

et al., 2007).

3. The 3 end of some viral RNAs does not perform a

special function in initiation of translation

3.1. TYMV

The hypothesis proposed by Barends et al. (2003) and

refuted by Matsuda and Dreher (2007) is that a tRNA-like

sequence at the 3 end of TYMV mRNA, which can be charged

with valine, reaches back to the 5 end of the mRNA and

195M. Kozak / Gene 403 (2007) 194203


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functions instead of Met-tRNAi to initiate translation from the

second AUG codon, producing a 206 kDa polyprotein. In

principle, there is no need for such a fancy mechanism to explain

translation of the polyprotein. The first AUG in TYMV mRNA,

which initiates translation of a 69 kDa movement protein, is in a

weak context which should allow leaky scanning; and there is

indeed evidence that some ribosomes gain access to the secondAUG via leaky scanning (Matsuda and Dreher, 2006).

As evidence for the reachback hypothesis, Barends et al.

(2003) claimed that [3H]valine was donated from the 3 tRNA-

like element to polypeptide(s) synthesized in a cell-free

translation system programmed with TYMV mRNA. The

main product labeled by [3H]valine was said to be a fragment

of the TYMV polyprotein, although its molecular weight was

different (19 kDa) and the identification was not confirmed by

immunological techniques. Thus, the evidence was weak to

begin with. A followup test in which the incorporation into

proteins of viral-linked radiolabeled valine was suppressed in

the presence of free unlabeled valine (Matsuda and Dreher,2007, Fig. 4) makes it unlikely that valine really is donated

directly from TYMV RNA into viral proteins.

As a second line of evidence, Barends et al. (2003) examined

[35S]methionine-labeled proteins directed by TYMV mRNA

which had been pre-treated with RNase H to remove the 3

tRNA-like element. The authors say the truncated mRNA

specifically lost the ability to synthesize the polyprotein; but the

evidence was not convincing because (i) the 206 kDa protein

was difficult to distinguish from other bands in the gel, and (ii)

there was no proof that RNase H cut only at the intended sites in

the 3 UTR. When the point was retested by Matsuda and

Dreher (2007) using antisera to identify the polypeptides and

avoiding the use of RNase, deletion of the 3 tRNA-likestructure had no effect on translation of viral proteins in vitro.

In formulating the reachback model, Barends et al. (2003)

ignored their own finding that oxidation of the 3 cytosine in

TYMV mRNA, which obviously precludes aminoacylation,

caused no reduction in synthesis of viral proteins in vitro. Thus,

the accumulated evidence strongly contradicts the reachback

mechanism.

3.2. Rotavirus

The closed-loop model postulates that contact between poly

(A) binding protein (PABP) and eIF4G augments translation.This caused people to wonder how non-polyadenylated

rotavirus mRNAs get translated. One idea was that a

nonstructural viral protein (NSP3) might substitute for PABP,

based on the finding that NSP3 binds to both eIF4G and a

sequence near the 3 end of rotavirus mRNAs (Piron et al.,

1998). Vende et al. (2000) argued that this binding has functional

consequences; i.e. they say circularization of rotavirus mRNAs

by NSP3 is necessary for efficient translation. Their experiments

employed unnatural systems, however; e.g. translation of

uncapped mRNAs in vitro, and translation of chimeric (rather

than authentic viral) mRNAs in a cell line that expresses NSP3

constitutively. Other deficiencies in these experiments were

explained in an earlier review (Kozak, 2004).

The notable new finding derived from straightforward

measurements of viral protein synthesis in infected monkey

cells is that siRNA-mediated knockdown of NSP3 had no

detrimental effect on production of other rotavirus proteins.

Thus, Montero et al. (2006) concluded that NSP3 does not

augment translation of rotavirus mRNAs.

Not yet settled is the question of whether NSP3 inhibits hostprotein synthesis (perhaps by binding to eIF4G). Montero et al.

(2006) noted that synthesis of host proteins persisted, to some

extent, when NSP3 was knocked down.1 Piron et al. (1998)

emphasized a temporal correlation between formation of

eIF4G-NSP3 complexes and disruption of eIF4G-PABP com-

plexes (detected by immunoprecipitation), but there was a

temporal discrepancy in that the reduction in host protein

synthesis preceded the disruption of eIF4G-PABP complexes.

Host translation was markedly reduced by 3 h post-infection,

while the reduction in eIF4G-PABP complexes did not begin

until 4.5 h and was not complete until 6 h.

If the old hypotheses about rotavirus translation are wrong(Piron et al., 1998; Vende et al., 2000), what mechanisms operate

instead? My guesses are that (i) the mechanism of initiation of

translation is the same for host and viral mRNAs. The fact that

rotavirus mRNAs lack a poly(A) tail is irrelevant because PABP

does not really augment translation of other mRNAs. [This

problematic point is discussed in other reviews (Kozak, 2004,

2006).] (ii) eIF4G is required for translation of both host and

viral mRNAs; i.e. the normal cap-dependent mechanism

operates. The finding that knock-down of eIF4G caused no

reduction in translation of viral or host mRNAs (Montero et al.,

2006, Fig. 7) simply means that eIF4G is not limiting; the

residual amount of eIF4G (10%) is sufficient to support protein

synthesis. (iii) The eventual decline in host protein synthesiscould be explained by competition from the massive amount of

viral mRNAs produced in infected cells.

4. Misunderstandings about IRESs

4.1. Revised thinking about poliovirus neurotropism

The ability of wild-type but not vaccine strains of poliovirus

to cause paralytic disease, and the finding that key mutations

map to the 5 UTR in Sabin vaccine strains, made it reasonable

to think that poliovirus neurotropism might be determined by

cell-type specific translation.2

Many experiments were inter-preted as evidence for this idea, which now appears to be wrong.

Recent experiments rule out tissue-specific translation as an

1 An experiment thatused firefly luciferase (Fluc) as a stand-infor host mRNAs

is suspect, however, because the cells were transfected withthe Fluc plasmid after

the onset of infection. The strong inhibition of Fluc expression, except when

NSP3 was knocked down, might reflect a block at the level of mRNA processing

or transport which would not apply to the pool of cellular mRNAs that existed

prior to infection.2 The 5 UTR of picornaviruses is involved also in RNA replication and

packaging (Borman et al., 1994; Hunziker et al., 2007; Johansen and Morrow,

2000), and therefore a defect at the level of translation is not the only way toexplain the effects of the mutations.

196 M. Kozak / Gene 403 (2007) 194203


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explanation for why poliovirus replicates predominately in the

brain and spinal cord.

The new experiments involve testing the ability of the

poliovirus IRES to support translation in mice, using an

adenovirus vector to express a dicistronic mRNA in which a

wild-type or mutated IRES precedes the 3 Renilla luciferase

(Rluc) reporter gene (Kauder and Racaniello, 2004).3 Thisapproach ensures that other effects of the mutations (e.g. on

RNA replication) are not mistaken for effects on translation. The

most important finding was that the poliovirus 5 UTR supports

translation, not only in the brain and spinal cord, but in many

other organs. The authors conclude also that a translational

defect associated with the Sabin type 3 IRES was observed in all

organs. (That conclusion requires qualification. With dicis-

tronic constructs containing the Sabin-3 mutation, production of

Rluc was reduced 3-to 5-fold compared to the wild-type IRES;

but the absence of RNA analyses makes it hard to know if the

primary effect of the mutation was on translation or mRNA

accumulation. Either way, there clearly was no organ-specificeffect.)

If it is not true that neurovirulent (wild type) poliovirus is

translated better in neuronalcells than in other tissues,and not true

thatSabin strains are attenuated specifically in neuronal cells, how

did those mistaken ideascome to be believed?Re-reading some of

the early papers is informative. One study (Agol et al., 1989)

admits to screening three neuronal cell lines and then proceeding

with the line that best discriminated between neurovirulent and

attenuated strains. (An addendum mentions that not all

neurovirulent strains grow as well, and not all attenuated strains

grow as poorly, as the ones used in the published experiments).

Another study says we sought a cell line in which replication of

attenuated poliovirus would be impaired (La Monica andRacaniello, 1989; the chosen line was SY5Y). As years passed,

people forgot about this biased screening as they clung to the

conclusion that there is a correlation between mutations in the

IRES and restricted growth in neuronal cells.

What generated the mistaken belief that a deficiency at the

level of translation underlies the purportedly restricted growth of

vaccine strains in neuronal cells? The aforementioned study with

SY5Y cells in which the Sabin-3-like virus replicated poorly

(ten-fold less efficiently than wild-type poliovirus; La Monica

and Racaniello, 1989) showed only a two-fold reduction in

synthesis of viral proteins; this was cited as evidence of impaired

translation of the attenuated virus. In later studies, thesupposedly impaired translation in neuronal cells was blamed

on impaired binding of polypyrimidine tract binding protein

(PTB), although binding of PTB to the IRES was barely

detectable even with wild-type poliovirus (Guest et al., 2004;

Gutierrez et al., 1997). Other evidence was obtained using in

vitro translation systems, which can be manipulated to show

almost anything. In one study, Haller et al. (1996) tested the

effects of adding an extract from neuronal cells to a reticulocyte

translation system programmed with RNA from laboratory-

constructed mutant viruses (revertants of X472) that purportedly

mimic Sabin-1. Their main finding was that a crude extract from

NGP neuronal cells, which stimulated translation of wild-typepoliovirus, fai led to stim ulate translation of the X472

revertants.4 If Kauder and Racaniello (2004) are correct, all

these hints of tissue-specific translational defects are artifacts

worth talking about only as warnings about similar experiments

with other putative IRESs.

4.2. Unjustified claims about cellular IRESs and disease

Experiments with a mouse model of dyskeratosis congenita

(DC) gave rise to the idea that translation is impaired by

mutations in the DKC1 gene, which encodes an enzyme

(dyskerin) that modifies uridine residues in rRNA. The claimthatDkc1m cells are specifically impaired in translation of IRESs

(Yoonet al., 2006) requires strongevidence that thethree affected

mRNAs Bcl-xL, p27Kip1 and X-linked inhibitor of apoptosis

(XIAP) are translated by internal initiation, but that key point

was not proven. Earlier evidence for IRES activity in XIAP and

p27Kip1 mRNAs did not hold up; re-analysis uncovered cryptic

promoters and splicing (Liu et al., 2005; Van Eden et al., 2004a).

Ignoring those problems, Yoon et al. (2006) argue that their claim

is supported by the finding that XIAP expression is stimulated by

irradiationin normalbut notDkc1m mutantcells; i.e. they argue

(unconvincingly) that stimulation by irradiation is proof of

IRES-dependent translation.

Inasmuch as Bcl-xL had not previously been identified as anIRES, it was crucial to test that point, e.g. by RNA transfection

experiments with dicistronic (5-Rluc-Fluc-3) transcripts. This

crucial test had no controls, however (Yoon et al., 2006, Fig. 2G).

The Bcl-xL IRES was compared only to itself(i.e. the Fluc/Rluc

ratio in normal unirradiated cells was set at 1.0 without revealing

how the Fluc yield from the Bcl-xL dicistronic construct

compares to a monocistronic mRNA, or a dicistronic mRNA

lacking an IRES, or a dicistronic mRNA containing a proven

IRES, if such there is). The argument used here has a circular

quality: the slightly reduced translation of Bcl-xL dicistronic

constructs in Dkc1m cells (compared to normal cells) is cited as

evidence that Bcl-xL is an IRES, and reduced translation of Bcl-xL is then cited as evidence that Dkc1m cells are impaired in

translation of IRESs.

In short, the studies with mouse Dkc1m cells are unconvinc-

ing; and the underlying idea of a ribosomal defect in Dkc1m

3 The claim that the poliovirus sequence indeed functions as an IRES in these

constructs was not proven rigorously. The proffered Northern blot is not

sufficient to rule out a small amount of monocistronic mRNA, perhaps produced

via a splice-donor sequence in the adenovirus portion of the vector and a splice-

acceptor sequence in the poliovirus IRES. The negative control (5NC-X472)

was not included in the Northern blot, and therefore it is possible that the

diminished yield of Rluc from that construct reflects diminished production ofmRNA rather than inactivation of the IRES.

4 The conclusion from these convoluted in vitro experiments was contra-

dicted by a straightforward experiment wherein viral protein synthesis was

monitored in NGP cells infected with Sabin-1 or wild-type poliovirus: there

was no difference between the two viruses. Haller et al. (1996) say this

unexpected result increases the value of experiments using the X472 revertants

because these viruses have neuronal cell-specific defects in translation that are

even more pronounced than those of the Sabin type 1 attenuated strain.

Readers might disagree about the value of substituting laboratory constructs forthe real thing.

197M. Kozak / Gene 403 (2007) 194203


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cells is contradicted by studies with human cells. Instead of a

ribosomal deficiency, cells derived from DC patients were

found to be deficient in telomerase RNA (Wong and Collins,

2006).

Another recent study postulates a defect in internal initiation

as a step in tumorigenesis (Wilker et al., 2007). The hypothesis is

that a protein called 14-3-3, which is frequently lost in tumors,normally stimulates IRES-mediated translation of the cyclin-

dependent kinase PITSLRE. The authors assumed, without

retesting, that PITSLRE is translated via an IRES. [Although

early experiments suggestive of internal initiation looked

convincing (Cornelis et al., 2000), the apparent IRES activity

was much lower in a follow up study (Tinton et al., 2005). Thus,

this essentialpoint requires verification.] In support of their claim

that IRES-dependent translation is impaired in cellslacking 14-3-

3, Wilker et al. (2007) show experiments carried out only with

the dubious p27Kip1 IRES (Liu et al., 2005) and an even more

dubious IRES from human immunodeficiency virus (HIV).5 The

bottom line is that it might be true that the aberrant phenotype of14-3-3-depleted cells results fromabsence of a short form (p58)

of PITSLRE, but p58 is probably translated from a spliced

mRNA (Xiang et al., 1994) rather than via internal initiation.

4.3. False deductions about the frequency of internal initiation

Faulty reasoning underlies the prediction that a sizeable

proportion of cellular mRNAs, perhaps as much as 35%is

likely to contain an IRES (Holcik and Sonenberg, 2005). That

number is based on the finding that 35% of cellular mRNAs

remain polysome-associated in poliovirus-infected cells, wherein

eIF4G undergoes cleavage (Johannes et al., 1999). But the belief

that there is a link between cleavage of eIF4G and inhibition ofcap-dependent protein synthesis is not well-founded: it is based on

correlative evidence from infected HeLa cells; but in some other

cell lines, host protein synthesis persists at normal levels even

after nearly-complete cleavage of eIF4G (Yanagiya et al., 2005;

5 hr time point).6 In one system where experimentally-induced

cleavage of eIF4G did reduce protein synthesis, the residual

translation was traced to localization of certain mRNAs in the

endoplasmic reticulum rather than to a special cap-independent

mechanism of translation (Lerner and Nicchitta, 2006).

The disconcerting absence of shared or similar sequences

among putative IRESs (Baird et al., 2006) is sometimes excused

by postulating that IRESs might instead have a common

secondary structure. Baird et al. (2007) undertook a search for

new IRESs based on that idea. But the search started from a

flawed premise the hypothetical folded structure proposed for

the XIAP IRES turned out not to be important for function

and the search uncovered no evidence for a common structure

among cellular IRESs. The absence of structural criteria thus

allows almost anything to be called an IRES (e.g. Dorokhovet al., 2002; Terenin et al., 2005).

Re-examination of many putative IRESs revealed cryptic

promoters or splice sites which undermine use of the dicistronic

test for internal initiation (Bert et al., 2006; Elango et al., 2006;

Kozak, 2005; Wang et al., 2005). This has taught editors to

scrutinize manuscripts more carefully, but the scrutiny still is not

careful enough. In some cases where authors say cryptic promoters

were ruled out, the test was not performed properly.7 The examples

in Table 1 reveal other ways of failing to prove while claiming to

prove that dicistronic vectors function as intended. The simplest

way for editors to demand more stringent criteria would be to ban

use of the popular pRF vector. The finding that detection of IRESactivity (even with poliovirus RNA) depends on the arrangement

of reporter genes in this vector (Hennecke et al., 2001) isa warning

that should not be ignored. The simplest explanation is that the

5 Rluc gene harbors a splice-donor sequence (verified by Van

Eden et al., 2004a) and the putative IRES actually functions by

contributing a splice-acceptor sequence.

There is growing use oftwo new indirect assays which do not

really rule out splicing. Van Eden et al. (2004a) say IRES activity

can be verified and splicing ruled out by demonstrating that

siRNA against the 5 Rluc cistron causes equivalent reduction of

Fluc expression from the 3 cistron. But the fact that Fluc was

resistant to inhibition by siRNARluc in one case where Fluc was

translated from a spliced mRNA (Van Eden et al., 2004a, Fig. 1E)does not necessarily mean that splicing can be ruled out in every

case where Fluc expression is inhibited by siRNARluc. The timing

of events (splicing vs. cleavage by siRNA) might vary from case

to case, and therefore the effect of siRNARluc on Fluc cannot be

predicted. The second assay does not involve siRNA; it simply

requires quantifying the mRNA derived from each cistron. Holcik

et al. (2005) say splicing can be ruled out if there is no

diminishment in the amount of RNA from the 5 cistron, reasoning

that production of a spliced transcript that preserves the 3 cistron

requires eliminating the 5 cistron. Although this subtraction

assay indeed detected the efficient splicing of Rluc/Fluc

transcripts caused by the putative XIAP IRES, the subtractionassay is not likely to detect a small amount of splicing which

would be sufficient to give the appearance of weak IRES activity.

In short, a positive result using one of these indirect assays might

indicate the occurrence of splicing, but a negative result does not

rule it out.

Advocates invoke indirect evidence when attempting to

prove the occurrence of internal initiation, but they overlook the

implications of indirect evidence when it works against them.

5 The putative IRES from HIV barely functioned when tested in vitro by

Brasey et al. (2003). It was more active when dicistronic constructs were tested

in vivo, but there were no RNA analyses to rule out splicing, and the IRES

activity inexplicably depended on using Fluc as the reporter. Wilker et al.

(2007) describe their own results with the HIV IRES in a way that hides the

inefficiency: the Fluc/Rluc ratio is simply set at 1.0 in control cells prior to

onset of mitosis.6 This might be because eIF4G is normally present in excess (as suggested by

the knockdown experiment in Montero et al., 2006), or because eIF4G cleavage

fragments can still support cap-dependent translation (Ali et al., 2001), or

because the observed cleavage of eIF4G occurs during cell lysis rather than in

vivo. Invoking a second form of eIF4G (eIF4GII) is not a convincing

explanation for the continued translation, given the low abundance of thatisoform.

7 Instead of deleting the SV40 promoter from the dicistronic pRF vector, the

putative IRES was transposed to a promoterless monocistronic vector (Petz et al.,

2007; Yang et al., 2006). This is not valid because enhancer elements in the

dicistronic construct might be needed to activate a cryptic promoter (Bert et al.,2006).

198 M. Kozak / Gene 403 (2007) 194203


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I refer to the repeated discovery of splicing factors that stimulate

one or another putative IRES (Bedard et al., 2007; Bushell et al.,

2006; Holcik et al., 2003; Lin et al., 2007). This might be

grounds for suspecting that the putative IRESs really work via

production of spliced transcripts.

5. Misunderstood tools invite more misunderstandings

5.1. Some viral IRESs (if they are IRESs) are too weak to serve

as controls

There is growing use of putative IRESs from cricket

paralysis virus (CrPV) and hepatitis C virus (HCV) as tools to

evaluate other candidate IRESs (Li et al., 2006) and to probe

other ideas about translation (Colon-Ramos et al., 2006;

Gorgoni et al., 2005; Humphreys et al., 2005; Petersen et al.,

2006). This is unwise, given the poor function and uncertain

mechanisms of these viral RNAs.8

The CrPV IRES works only 2% as efficiently as a normalcapped mRNA (Humphreys et al., 2005). The HCV IRES also

supports only low-level translation of the 3 cistron when

dicistronic mRNAs are tested in vitro (e.g. Miyakawa et al.,

2006, Fig. 4B; Van Eden et al., 2004b, Fig. 2B). When tested by

RNA transfection, dicistronic mRNA containing the HCV IRES

supported only threefold better translation of the 3 cistron than a

control with no IRES (Van Eden et al., 2004b). [The HCV IRES

appears to work better when tested by DNA transfection, but that

can be attributed to cryptic promoter activity (Dumas et al.,

2003).] In many studies, the poor performance of the HCV or

CrPV IRES is concealed by setting the yield at 1.0 without

reference to any other mRNA (e.g. Berlanga et al., 2006; Coller

and Parker, 2005; Colon-Ramos et al., 2006; Gorgoni et al.,2005; Holz et al., 2005; Lancaster et al., 2006; Moerke et al.,

2007; Petersen et al., 2006; Yoon et al., 2006).

5.2. Edeine does not inhibit polypeptide elongation

Experiments in which edeine is used to distinguish between

standard and special initiation mechanisms often are misinter-

preted. This is an important issue in evaluating claims about

CrPV. The undisputed finding is that translation of CrPV is

inhibited by edeine at a concentration of 2 M (Matsuda and

Dreher, 2007, Fig. 2B; Wilson et al., 2000, Fig. 3K). When

people want to argue that a special edeine-resistant mechanismof initiation operates, however, they dismiss that result by

claiming a high concentration of edeine (e.g. 2 M) inhibits the

elongation step. That simply is not true. Edeine is used

routinely at a concentration of 2 or 5 M to inhibit initiation and

thus allow study of the elongation step (Fang et al., 2004;

Horsburgh et al., 1996; Mothes et al., 1997; Somogyi et al.,

1993). In a classic experiment undertaken to test the point

directly, Hunt (1974) showed that 2 M edeine inhibits

initiation and not elongation of globin polypeptide chains.The unexpected finding that CrPV-mediated translation is

not inhibited by lower concentrations of edeine (e.g. 0.5 M)

might be an artifact caused by the low efficiency of the putative

IRES: translation is probably limited, not by the availability of

ribosomes, but by the small amount of functional (broken)

mRNA. Not until the entire pool of ribosomes is inactivated

(at 2 M edeine) would translation of CrPV be affected.

Lancaster et al. (2006) defend the idea that edeine inhibits

elongation by citing studies by Szer and Kurylo-Borowska

(1970) and Carrasco et al. (1974). The first of these papers reports

that edeine inhibits elongation in a bacterial system, which is not

relevant to how the drug works in eukaryotes. The second papertested effects of edeine using poly(U) as the template, which is

not relevant to how the drug works with natural mRNAs.

5.3. Indirect tests and blind use of reagents contribute to

misunderstandings about microRNAs and IRESs

A precision tool should not be used like a hammer. It is

worrisome to see reports wherein rapidly-sedimenting com-

plexes are judged to be polysomes based on their disappear-

ance from cells exposed for a very long time (3 h) to a very high

concentration of puromycin (1 mM) (Vasudevan and Steitz,

2007). This exceeds the conditions routinely used to dissociate

polysomes [e.g. incubation of HeLa cells with 200 Mpuromycin for 45 min (Katze et al., 1986)], and even those

conditions are excessive. Nottrott et al. (2006) found nearly

complete release of mRNAs from polysomes after cells were

incubated with 5 M puromycin for only 20 min.

In another attempt to study the mechanism of inhibition by

microRNAs, Thermann and Hentze (2007) used a crude

translation system from Drosophila embryos which they say

works only if the reporter mRNA is pre-incubated in the extract

for three hours. They say nothing about what happens during

that long interval. The experiments can be faulted on many

other grounds, e.g. the rapidly-sedimenting pseudo-poly-

somes, purportedly induced by microRNA, were seen evenunder conditions where translation was not repressed. But it

seems trivial to fuss about such problems when the experiment

begins with a three-hour black box step.

IRESs are used as tools in many studies with microRNAs.

The reasoning is that, if IRES-mediated translation is not

inhibited, microRNAs must inhibit via the cap. The test is

indirect, at best; but it loses all meaning when the putative IRES

barely works [the CrPV IRES was only 2% as efficient as

capped mRNA in the study by Humphreys et al. (2005)] and

when the answer varies depending on the transfection technique

(Lytle et al., 2007).

IRES activity itself is sometimes claimed based only on

indirect tests; e.g. demonstrating that translation is cap-

8 Advocates claim to have proven that the CrPV IRES forms a functional

complex with ribosomes; i.e. they claim the complexes (assembled without

initiation factors and Met-tRNAi) go on to produce peptides or peptidyl-

puromycin (Cevallos and Sarnow, 2005, Fig. 8; Jan et al., 2003; Pestova and

Hellen, 2005). But those experiments were not convincing because there was

no positive control against which to judge the efficiency of the reaction with

CrPV mRNA. Without convincing evidence of function, elegant structural

models cannot be interpreted. The need to use yeast ribosomes and a high

concentration of Mg

2+

to obtain stable complexes with CrPV RNA (Schuleret al., 2006) adds to the worry about what the structural models really mean.

199M. Kozak / Gene 403 (2007) 194203


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independent (not inhibited by a cap analog; Marr et al., 2007) or

not inhibited when a hairpin structure is appended to the 5 end

of the mRNA. These tests are meaningless because translation

from broken transcripts also would be unaffected by those

manipulations. Broken transcripts that lack a cap cannot be

translated efficiently, but they can be translated (Bendena et al.,

1985; Kozak, 1980; Lawrence, 1980; Pelham, 1979; Thoma

et al., 2001). Thus, low-level translation of the 3 proximal

cistron might be accomplished, not via internal initiation, but

via cleavage of dicistronic mRNAs.9

Possible cleavage of mRNA is an important issue vis--vis

interpreting experiments with both IRESs and microRNAs.Investigators often try to address this concern by showing that

intact transcripts can be recovered following liposome-mediated

transfection, but the recent finding thatmostof the RNA introduced

via liposomes does not enter the cell in a usable way (Barreau

et al., 2006) precludes simple conclusions. When the structure and

stability of the functional mRNA pool is at issue, analysis ofbulk

mRNA recovered from transfected (or microinjected) cells is

meaningless. Rulingout effects on mRNA stability can be difficult

even when translation is studied in vitro, as in a recent attempt to

study inhibition by microRNAs using extracts from Krebs cells

(Mathonnet et al., in press). Sucrose gradient analysis of initiation

complexes revealed that only 10% of the input mRNA actually

participates in translation; and therefore, the absence of an effect

on bulk mRNA stability does not rule out the possibility that

microRNAs inhibit by cleaving (translationally active) mRNAs.

6. Last thoughts

Some problems are becoming more common as investigators

(mis)learn from one anothercopying poor experimental

designs (e.g. dicistronic vectors); repeating erroneous inter-

pretations (e.g. that edeine inhibits elongation); predicating new

models on dubious old models. The mistaken idea about

translation of TYMV (Section 3.1) was inspired by a dubious

model about Met-tRNAi-independent translation of CrPV. The

mistaken idea about translation of rotavirus mRNA (Section 3.2)

was inspired by the unproven closed-loop model.

A frequent problem is deciding the answer first and then

constructing a test system to show what one wants to see, as in

the selection of a neuronal cell line that showed the expected

9 Some picornavirus IRESs contain sequences that are specifically recognized

by RNases (Elgadi and Smiley, 1999; Serrano et al., 2007), but random attack is

probably the more common mechanism. Rosenfeld and Racaniello (2005)

claimed that cleavage of dicistronic mRNA was ruled out in their study because

translation of the 3 cistron was not reduced by mutations that inactivate the

nonsense-mediated decay pathway, but that pathway is not the only source of

RNases in yeast. In the case of HCV, cleavage might even occur bymagnesium-catalyzed autohydrolysis (Kieft et al., 1999).

Table 1

Recent poorly-documented claims of IRES activity in 5 UTR sequences from cellular mRNAs

mRNA Problems with the evidence obtained via dicistronic vectors a References

Cryptic promoter? Splicing checked? Activity confirmed via RNA

transfection?

XIAP (intron)b Ruled out. Splicing was detected with

pRF vectorcVery weak (twofold greater

than empty vector).

Van Eden et al. (2004a)

NF-B repressing factor (intron) b Ruled out. siRNA against 5 Rluc also

inhibits 3 Fluc.

Not tested. Reboll et al. (2007)

p27Kip1 Detected but

deemed minimal. dsiRNA against 5 Rluc also

inhibits 3 Fluc.

Absence of a positive control

precludes conclusion.eJiang et al. (2007)

AANAT f Ruled out. Northern blot is insufficient

to rule out splicing.

Not tested. Kim et al. (2007)

Cyclin T1, SET7, SIAH2, ZNF217, etc. Ruled out. Northern blot is insufficient

to rule out splicing. gNot tested. Bushell et al. (2006)

a The splicing-prone pRF vector (5-Rluc-Fluc-3) was used for all these studies. Section 4.3 explains why the use of siRNA, targeted to the 5 cistron, is not a valid

assay for ruling out production of spliced (monocistronic) mRNAs from the dicistronic vector.b cDNA analyses revealed that the long 5 UTR, here tested for IRES activity, is really an intron (Jianfeng et al., 2003). With XIAP also, the 3 end of the putative

IRES harbors a splice acceptor site that functions naturally, i.e. the IRES is an intron ( Van Eden et al., 2004a).c Holcik et al. (2005) claim that with a different dicistronic vector (gal/CAT), the XIAP sequence supports translation in the absence of splicing, judging from the

fact that there was no reduction in the amount ofgal RNA. But this experiment had no positive control to show that the predicted reduction in gal can be detectedwhen a verified intron is introduced.d The detected activity, equal to 15% of the strong SV40 promoter, is arguably more than minimal.e The efficiency of expression from the dicistronic transcript needs to be compared to a capped monocistronic mRNA (positive control). Claims of IRES activity

based only on comparison to a negative control are meaningless because it is too easy to devise a negative control (in this case, an antisense version of the p27Kip1 5

UTR) that makes the sequence of interest look good. In RNA transfection experiments with a different candidate IRES, Van Eden et al. (2004b, Fig. 5C) demonstrated

vastly different levels of expression of the 3 cistron with three different negative controls (random insertNempty vectorantisense insert). It makes no sense to use the

worst of these as the control against which IRES activity is calculated.f Insertion of the putative IRES from arylalkylamine N-acetyltransferase (AANAT) mRNA produced only a fourfold increase in translation of Fluc relative to the

empty pRF vector. Thus, the putative IRES is very weak, as shown also by comparing translational yields from the 3 and 5 cistrons (Flucb2% of Rluc).g Bushell et al. (2006) argue that Northern blotting, albeit insensitive, really is the best method to check RNA structure because RT-PCR fails when the wrong

primer is used. Internal initiation was proven, they say, by demonstrating that dicistronic mRNA is polysome-associated even when translation of the first cistron is

blocked; but the fine-print reveals the RNA analyses were done on fractions pooled from polysomal and 80S regions of the gradient, which makes the claim

meaningless.

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(wrongly expected) discrimination against vaccine strains of

poliovirus (Section 4.1). The problem of allowing expectations

to dictate the outcome can be seen also in a recent study of the

mechanism whereby translation of PRMT5 is elevated in

lymphoid cancer cells. Pal et al. (2007) decided a priori to

look for microRNAs targeted to PRMT5 mRNA, without having

ruled out simpler ways to explain the up-turn in translation; e.g.although they checked for (and ruled out) an increase in the

amount of mRNA, they did not check for a possible change in

structure of the 5 UTR.10 Upon finding that addition of the

candidate microRNAs to an in vitro translation system failed to

inhibit translation of PRMT5, the authors simply changed the

sequence of the microRNAs to make up for the lack of

stability.

The internal initiation story is the most egregious example of

deciding the answer first. Unlike the poliovirus vaccine story,

where people selected evidence that fit expectations, the

evidence for internal initiation was invented rather than selected.

This was not done in a fraudulent way, of course; it was done byadopting such weak criteria for recognizing IRES activity that

any sequence of interest could be called positive.11 The

starting belief (pre-decided answer) was that critical regulatory

genes e.g. genes expressed during mitosis or apoptosis need

a special mechanism for initiating translation (Holcik and

Sonenberg, 2005). This prompted investigators to test important

regulatory genes for IRES activity; and because of the weak

criteria (and use of the pRF vector), almost every test worked.

Advocates say the belief has been vindicated. Critics are still

watching for the first scrap of real evidence.

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