2014

http://informahealthcare.com/bmgISSN: 1040-9238 (print), 1549-7798 (electronic)

Editor: Michael M. CoxCrit Rev Biochem Mol Biol, 2014; 49(2): 164–177

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10409238.2014.887051

REVIEW ARTICLE

Transcriptome-wide studies uncover the diversity of modes of mRNArecruitment to eukaryotic ribosomes

Ivan N. Shatsky1, Sergey E. Dmitriev1,2, Dmitri E. Andreev1, and Ilya M. Terenin1,2

1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia and 2Engelhardt Institute of Molecular

Biology, Russian Academy of Sciences, Moscow, Russia

Abstract

The conventional paradigm of translation initiation in eukaryotes states that the cap-bindingprotein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in therecruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicatesthat this paradigm should be revised. This review summarizes the data which have been mostlyaccumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wideanalysis. Unexpectedly, these techniques have uncovered remarkable diversity in therecruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminaryclassification of mRNAs into several groups based on their requirement for particularcomponents of eIF4F. They challenge the widely accepted concept which relateseIF4E-dependence to the extent of secondary structure in the 50 untranslated regions ofmRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 50 ends withoutthe involvement of either the 50 m7G-cap or eIF4F but instead utilize eIF4G or eIF4G-likeauxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements incellular mRNAs is also discussed.

Keywords

50 UTR secondary structure, cap-independenttranslation enhancer, cellular IRESs, DAP5,eIF4F, eIF4G, ribosome profiling,translation initiation factor eIF4E

History

Received 4 November 2013Revised 17 January 2014Accepted 21 January 2014Published online 13 February 2014

Introduction

Translational control mainly operates at the initiation step of

polypeptide synthesis. The basis of translation initiation in

eukaryotes was elucidated in the 1970 and 80s, starting with

the discovery of the mRNA scanning mechanism by Kozak

(1989). This important event occurred in a ‘‘pre-genomic

era’’ when the set of available model mRNAs was extremely

limited and was not representative of the entire diversity of

mRNA structures. These ‘‘single mRNA’’ studies resulted in

a widely recognized model of cap-dependent translation

initiation which is believed to be used by the overwhelming

majority of cellular mRNAs in all eukaryotes. Somewhat later

(in the 1990s), an alternative mechanism – internal initiation

of translation – was discovered (Jang et al., 1988; Pelletier &

Sonenberg, 1988) and dissected (Pestova et al., 1996a,b,

1998) for uncapped genomic RNAs from several viruses

replicating in the cytoplasm. According to this mechanism,

the ribosome recognizes the mRNA at a specific internal

ribosome entry site (IRES) within its 50 untranslated region

(50 UTR). Whether this alternative mechanism of ribosome

recruitment to mRNA is also used by cellular mRNAs, in

particular by mammalian mRNAs, is still a matter of debate

(see below).

With the beginning of the transcriptome-wide era

(a discussion of respective techniques can be found in

Kapeli & Yeo, 2012), we now have the ability to analyze

large numbers of structurally diverse mRNAs while manip-

ulating specific key components of the cell or signaling

pathways. Some of the emerging data cannot be explained on

the basis of either the standard model of mRNA recruitment

to ribosomes or with the help of IRES-elements. These data

suggest that something very important has been missed in the

past, presumably since a large variety of mRNA structures

was not taken into account. Without filling these gaps in our

ideas on the molecular mechanisms of recruitment of various

mRNAs to ribosomes, we cannot advance our studies of the

mechanisms of translational control.

The aim of this review is to share our observations while

mining the literature of the last decade. We decided to focus

on the very first events of mRNA recruitment onto ribosomes

(40S subunits) and will not discuss the subsequent steps of

translation initiation (such as ribosomal scanning, start codon

selection or subunit joining) since these issues have been

covered in a number of comprehensive recent reviews

(Alekhina & Vassilenko, 2012; Hinnebusch & Lorsch, 2012;

Sonenberg & Hinnebusch, 2009;). We will not describe all

known cases of translational control, either. Instead we wish

to highlight those studies which do not easily align to the

Address for correspondence: Dr. Ivan N. Shatsky, PhD, BelozerskyInstitute of Physico-Chemical Biology, Lomonosov Moscow StateUniversity, Leninskie gory 1, Khokhlov str., bldg. 40, Moscow119234, Russia. Tel: +7495 939 4857. E-mail: shatsky@genebee.msu.su

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current paradigm of primary mRNA recognition by

ribosomes.

The conventional mechanism of mRNA recruitmentto ribosomes in eukaryotes and factors involved inthis process

The classical mechanism (Sonenberg & Hinnebusch, 2009)

states that translation initiation starts with the binding of

factor eIF4E to the cap of mRNA. In the mammalian cell,

eIF4E1 (a prevalent version of eIF4E) may be: (1) in a free

state, (2) in a complex with its repressor, eIF4E-Binding

Protein (4E-BP), or (3) part of an active heterotrimer, termed

as eIF4F (eIF4E�eIF4G�eIF4A). eIF4G plays the role of a

scaffold in the trimer eIF4F. This large multifunctional

protein (M.W. �175 kDa) interacts with several ligands

involved in the accommodation of mRNA on the 40S

ribosomal subunit (Figure 1A). For mammalian eIF4G,

these include interactions with: eIF4E (which holds the

mRNA by the cap), eIF3 (which bridges the complex to the

40S subunit), eIF4A (an ATP-dependent RNA helicase), and

PABP (Poly(A) Binding Protein) bound to the mRNA

poly(A)-tail. In addition, eIF4G can also bind the mRNA

itself, contacting not only its 50 UTR but in some cases the 30

UTR (Park et al., 2011a), by means of distinct RNA-binding

sites. Yeast eIF4G has at least three sites that interact with

mRNA (Berset et al., 2003; Park et al., 2011a), while

mammalian eIF4G has two known RNA-binding sites (Prevot

et al., 2003). No information is available about the nucleotide

sequence preferences of eIF4G for RNA binding.

All these interactions result in the mRNA pseudo-

circularization which is thought to reinforce interactions that

help keeping eIF4F on the mRNA. Indeed, the interaction of

eIF4G with PABP increases the affinity of eIF4E for the cap

(Borman et al., 2000). However, our knowledge about its

Figure 1. eIF4G and eIF4G-like protein families. (A) Some characterized representatives of eIF4G family. The identified structural domains aredesignated inside the boxes whereas the binding sites for initiation factors, PABP and Mnk1 and 2 kinases are shown above the diagrams. Thehorizontal arrows denote alternative translation starts in eIF4G1 of Homo sapiens. The horizontal brackets delimit either sequences missing in someisoforms or C-terminal truncated ones. The vertical long arrows under the boxes denote cleavage sites for picornavirus proteases 2A (2A-pro) and L(L-pro), caspase 3 (Casp3) and HIV-1/2 proteases. The sites of interaction with RNA are indicated by arrows above the diagrams. (B) MIF4G domaincontaining proteins that have been shown to participate in the mRNA recruitment to ribosomes. The binding sites for particular initiation factors areshown with the same color as in (A). The designations for conserved domains are: MIF4 for ‘‘Middle domain of eIF4G’’ (pfam02854); MA3 for‘‘Domain in DAP-5, eIF4G, MA-3 and other proteins’’ (pfam02847); W2 for ‘‘C-terminal domain of eIF4-gamma/eIF5/eIF2b-epsilon’’ (pfam02020);KH for ‘‘hnRNP K homology RNA-binding domain, type I’’ (pfam00013).

DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 165

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contribution to the translation of individual natural mRNAs,

i.e. possessing distinct 50 and 30 UTRs, is still very limited

(Kopeina et al., 2008; Park et al., 2011b).

The activity of helicase eIF4A is important for both

accommodation of the 43S preinitiation complex (43S PIC) at

the 50 end of mRNA and subsequent mRNA scanning assisted

by eIF4B (Parsyan et al., 2011). eIF4B is indispensable for

eIF4A functioning during scanning of the 50 UTRs that have

even minor base-pairings within their sequences (Dmitriev

et al., 2003). It is possible that the primary accommodation of

the scanning apparatus on some 50 UTRs that have highly

stable stems at their 50 termini is also promoted by additional

DEAD-box helicases, e.g. DDX3 (Ded1p in yeast) or

DDX9/RHA (for review, see Marintchev, 2013; Soto-Rifo

et al., 2012). Unfolding of especially stable stem-loops during

the scanning may also require DHX29 (Pisareva et al., 2008).

For some mRNAs, eIF4B may be substituted by its homolog

eIF4H (for review, see Parsyan et al., 2011).

The chain of interactions eIF4E–eIF4G–eIF3–40S is one

of the principal targets for translational regulation. Although a

direct interaction of eIF4G with eIF3 has not been found in

yeast, it may be mediated by some other translation compo-

nents, e.g. by eIF5 or eIF4B. Yeast also demonstrate a less

pronounced dependence on eIF4G for mRNA recruitment

in vitro, with eIF3 playing the major role (Jivotovskaya et al.,

2006; Mitchell et al., 2010). However, the importance

of eIF4G for increasing the rate and extent of mRNA

recruitment in yeast is nevertheless in no doubt (Mitchell

et al., 2010).

Regulation of eIF4F integrity by eIF4E-bindingproteins and mTOR signaling

It should be mentioned that though eIF4E and eIF4G form a

complex, their individual concentrations in proliferating cells

are quite different. eIF4E1 is a very abundant protein, and

even when its concentration in mammalian cells was

decreased 10-fold by RNA interference, no significant

reduction in global protein synthesis was observed

(Yanagiya et al., 2012). This high molar excess of eIF4E is

partially neutralized in cells by multiple specific repressors

4E-BPs (see below). Therefore, it is the ratio of eIF4E1 to

active (hypophosphorylated) 4E-BPs what should be con-

sidered (‘‘active’’ eIF4E1 concentration) rather than the

absolute concentration of the factor (Alain et al., 2012).

In higher eukaryotes, the global translational regulation of

eIF4E is thought to be based on 4E-BPs 1, 2 and 3. These

small proteins harbor the eIF4E-binding motif YXXXXL�(where X is any amino acid and � is a hydrophobic residue)

that interacts with the same dorsal surface of eIF4E respon-

sible for its interaction with eIF4G (Mader et al., 1995).

In a hypophosphorylated (active) state, 4E-BPs compete with

eIF4G for eIF4E binding to sequester eIF4E from the eIF4F

complex (for review, see Roux & Topisirovic, 2012).

On receiving a proliferative signal, these eIF4E repressors

are multiply phosphorylated by the complex C1 of mam-

malian Target Of Rapamycin kinase (mTORC1) and revers-

ibly inactivated. As a consequence, eIF4E is liberated,

associates with eIF4G-eIF4A (thereby forming eIF4F),

and directs the mRNA to the 40S ribosomal subunit.

mTORC1 also activates the ribosomal protein S6 kinases

p70S6K1/2, which subsequently phosphorylate eIF4B and

Programmed Cell Death 4 (PDCD4), an inhibitor of eIF4A

that is targeted to proteolysis after the phosphorylation

(for review, see Roux & Topisirovic, 2012). Although

direct effects of eIF4B modifications are open to question

(reviewed by Shagam et al., 2012), both eIF4B and PDCD4

phosphorylation are necessary in vivo for the manifestation of

mTOR activation effects (Dennis et al., 2012). mTOR can

directly bind eIF3 and positively affect the eIF4G–eIF3

interaction (Harris et al., 2006). Its indirect targets also

include eIF4G, translation elongation factor eEF2 and ribo-

somal protein S6. mTOR also controls the transcription of

rRNAs and tRNAs (Laplante & Sabatini, 2012). Such a

multiplicity of complex events caused by activation of this

huge protein kinase may diminish to some extent a direct role

of 4E-BPs in response to proliferative signals. It is important

to note that overexpression of a completely non-phosphor-

ylatable mutant of 4E-BP1 in cells inhibited translation of

a reporter mRNA by only �60% and overexpression of

wt 4E-BP1 inhibited it by only 20% (Mothe-Satney et al.,

2000). Similarly, modest effects of recombinant 4E-BP1

addition were also observed in vitro (Andreev et al., 2009;

Haghighat et al., 1995).

mTOR is a downstream effector in the PI3K/AKT pathway

that has been linked to cancer, obesity, diabetes, neuro-

degeneration and aging (Laplante & Sabatini, 2012).

A number of other signaling cascades that translate external

signals into the cell converge on this pathway, thus affecting

mTOR activity: these include special GTPases that sense

amino acid availability, receptors for growth factors

(e.g. insulin, EGF and interferons) or various molecules

responsible for energy supply. A detailed description of

mTOR complexes Q1 and Q2 and their functions, as well as

implication of other signaling pathways in translation regu-

lation can be found in (Laplante & Sabatini, 2012; Roux &

Topisirovic, 2012; Wang & Proud, 2011).

All eukaryotes express proteins that can repress the

eIF4E–eIF4G interaction thus regulating large number of

mRNAs. Examples in yeast are Caf20p and Eap1p with MWs

of 18 and 70 kDa, respectively (Cridge et al., 2010). Except

for the eIF4E interaction motif, they are quite distinct from

4E-BPs of higher eukaryotes. They interact with eIF4E while

being associated with 30 UTRs of the mRNAs they regulate.

It has been shown that Caf20p and Eap1p together control

translation of �1000 mRNAs and each repressor regulates

its own class of mRNAs. Apart from general regulators of

eIF4E activity, eukaryotic cells also contain eIF4E-binding

proteins that regulate translation of subsets of mRNAs.

Among them, it is worth mentioning Cup in Drosophila,

Maskin in oocytes of Xenopus laevis and Neuroguidin

in neurons (for references see reviews by Darnell &

Richter, 2012; Lasko, 2012; Richter, 2007). However, this

mode of regulation requires the formation of complexes

of eIF4E-binding proteins with additional protein factors

which in turn recognize specific RNA-motives within 30

UTRs of respective mRNAs. This leads to pseudo-

circularization of mRNA, and therefore resembles the afore-

mentioned mechanism of action proposed for yeast Caf20p

and Eap1p.

166 I. N. Shatsky et al. Crit Rev Biochem Mol Biol, 2014; 49(2): 164–177

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Large variations in eIF4E-dependence for variousindividual mRNAs

The current literature, as a rule, proposes only two modes of

mRNA recruitment in eukaryotes – the classical mechanism

just described and the cap-independent IRES-driven mode of

initiation (Figure 2A and B). Until recently, no other

mechanisms of cellular mRNAs binding with 40S ribosomal

subunits were known. For the classical mechanism, the

interaction of the cap with eIF4E (mostly its eIF4E1 isoform

in mammals) is regarded as an obligatory first step in

recruitment of mRNAs to 40S ribosomes for the overwhelm-

ing majority of cellular mRNAs. Nevertheless, there is a

general agreement that the eIF4E dependence varies among

individual mRNAs, as some mRNAs still continue to bind

ribosomes under stress conditions when eIF4E1 is inactivated

by 4E-BPs and the scaffold factor eIF4G is cleaved by virus

proteases or caspases (Figure 1A). Such stress-resistant

cellular mRNAs are believed to harbor IRES-elements

within their 50 UTRs (for review, see Ruggero, 2013).

Conversely, another subset of mRNAs is believed to have

an increased requirement for eIF4E. Until recently this

property has been ascribed to those mRNAs that have long

and highly structured 50 UTRs (Gingras et al., 1999) and this

opinion still predominates (for references, see Jia et al., 2012).

Originally it came from two well-known reports where

mRNAs containing artificial perfect stem-loops of different

length in their 50 UTR were shown to require more eIF4E and

eIF4A for efficient translation (Koromilas et al., 1992; Svitkin

et al., 2001). Indeed, it is logical to assume that the structured

50 UTRs are more dependent on eIF4E since they may need

more helicase eIF4A for unwinding their stem-loop structures

and eIF4A is delivered to the 50 terminus through the

eIF4E�eIF4G complex. The recent unexpected finding that

eIF4E binding to eIF4G not only brings the complex to the 50

cap, but also stimulates in vitro the eIF4A-helicase activity in

a cap-independent manner, which has been presented as a fact

in favor of this hypothesis (Feoktistova et al., 2013). This is an

important point since highly structured 50 UTRs are charac-

teristic of many mRNAs encoding oncogenic growth and

transcription factors or regulatory protein kinases and these

considerations have been employed to explain why many

cancer cells (�30%) have elevated levels of eIF4E. The same

arguments have been used to explain why some specific

mRNAs with highly structured 50 UTRs are activated after the

knock-out of 4E-BPs in mice (Colina et al., 2008; Gkogkas

et al., 2013). Nevertheless, this concept should be revised: as

we shall see below, recent transcriptome-wide data do not

support it.

The initial testing of the correlation between the eIF4E

dependence and the length and degree of secondary structure

in natural 50 leaders was performed in 2009 (Andreev et al.,

2009). The analysis was carried out both in a cell-free system

and in RNA-transfected cells. It should be emphasized that for

in vitro assays a cytoplasmic extract from cultured mamma-

lian cells was used since the conventional rabbit reticulocyte

lysate was shown to be inadequate for translation initiation

studies of mRNAs possessing complex 50 leaders (Dmitriev

et al., 2009). Similarly, the RNA transfection technique was

chosen to avoid artifacts associated with DNA-encoded

reporters (Dmitriev et al., 2007; see also below). This study

showed that the 50 cap stimulated translation of mRNAs with

short and less structured 50 UTRs much more than some

mRNAs with long and highly structured 50 leaders. These

results were confirmed further by in vitro experiments

with 4E-BP1. Among the tested mRNAs, the lowest cap-

dependence, and therefore eIF4E-dependence, was the 50

UTR of Apaf-1 mRNA which possesses four highly structured

domains (though no IRES activity was detected within its

sequence) (Andreev et al., 2009).

Transcriptome-wide identification of mRNAs withincreased requirement for eIF4E

What classes of natural mRNAs are particularly sensitive to

the ‘‘active’’ eIF4E concentration? Or do 4E-BPs regulate

global protein synthesis? Of earlier reports on this point one

should mention the paper by (Mamane et al., 2007) where the

authors carried out a microarray analysis of polysomal mRNA

from an eIF4E-inducible NIH 3T3 cell line. Among 294

transcripts that shifted upon eIF4E induction to the heavier

polysome fractions, were mRNAs coding for components of

the translational machinery (ribosomal proteins, translation

factors, etc.) These mRNAs are known to possess relatively

short and low structured 50 UTRs.

The decisive contribution to clarifying this issue has been

done by recent transcriptome-wide studies by Thoreen et al.

(2012) and Hsieh et al. (2012). These authors treated mouse

p53 �/� embryonic fibroblasts (MEF) or human p53 �/�prostate cancer cells PC3, respectively, with inhibitors of the

ATP binding site of mTOR, thorin 1 and PP242. Both drugs

inhibit the catalytic subunit of mTOR and activate 4E-BP’s

(see above) much more efficiently than the well-known drug

rapamycin that acts allosterically. Changes in the translational

activity of individual mRNAs were assessed by ribosome

profiling, a novel powerful method of genome wide analysis

of mRNA activities in living cells (Ingolia et al., 2012).

A positive aspect in the design of both studies was a

short-time interval between the drug addition and the

moment when the cells were harvested, lysed and used in

ribosome profiling. In other words, the authors analyzed an

immediate response. This is an obvious advantage over the

experiments with 4E-BP knockout murine cells (see above)

where essential adaptive changes during development of

knockout murine stem cells (MSC) into embryos cannot be

excluded.

Surprisingly, the inhibition of eIF4E activity resulted in a

strong suppression of translation (from 4-to-8-fold) of only

120–150 mRNAs. The authors confirmed that this inhibition

was largely due to dephosphorylated 4E-BPs. The affected

mRNAs mostly coded for ribosomal proteins, translation

factors and proteins indispensable for growth rather than

oncogenic kinases, receptors or transcription factors. In fact,

this result is not as surprising as it seems at first glance: one of

the principal hallmarks of cancer cells is their enhanced

ability to grow and proliferate and mTOR is the one of the

main downstream regulators that governs these features. One

should bear in mind that growth and proliferation first

necessitate the synthesis of more ribosomes and translation

factors since the translational capacity of any cell is limited

DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 167

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168 I. N. Shatsky et al. Crit Rev Biochem Mol Biol, 2014; 49(2): 164–177

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and determined to a large extent by translation apparatus

availability. Conversely, mTOR inhibition immediately stops

the formation and function of protein synthesizing factories as

the most resource and energy consuming process. That is why

mTOR activity is linked to all or almost all other signal

pathways sensing environmental changes. In the case of

prostate cancer PC3, a strong decrease of translation was also

noted for four mRNAs that determined mesenchymal charac-

teristics of PC3 cells and their capability of metastasizing

(vimentin, YB-1, MTA1, CD44) (Hsieh et al., 2012).

Remarkably, the 50 UTRs of the majority of these highly

eIF4E-dependent mRNAs did not demonstrate an increased

length and/or GC-content. Just the opposite, they revealed

some slight bias to a shorter length and a lower base-pairing.

Their common distinctive feature was the presence of a 50

terminal oligopyrimidine tract (50 TOP). This feature is

known to determine the ease with which mRNAs are recruited

to polysomes after transferring starved cells to rich medium.

Strikingly, the authors found no more than 30% of the total

number (about 5000) of examined individual mRNAs which

were sensitive to some extent to mTOR inhibition. The

translation of other mRNAs either did not change at all or

were even stimulated, presumably due to less competition for

ribosomes from the very abundant mRNAs encoding compo-

nents of the translational apparatus. Among the stimulated

mRNAs there were some that mediate cell survival after drug

treatment (XIAP, Bcl2), proliferation (c-Myc) or apoptosis

(Apaf-1). All these transcripts have long and highly structured

50 UTRs but do not pass through stringent criteria for IRES-

containing mRNAs (reviewed by Shatsky et al., 2010, see also

below). Thus, translation of the majority of mRNAs in the

investigated cells depended very little on the concentration of

‘‘free’’ eIF4E1, and is unlikely to be directly controlled by

mTOR kinase. Moreover, the activity of some of them,

e.g. the stress-induced translation of Hsp70 mRNA, has been

reported to be negatively regulated by PI3K–mTORC1

signaling (Sun et al., 2011).

As the function of eIF4E in the mRNA recruitment into

48S complex may be realized exclusively via its interaction

with eIF4G, it is logical to assume that the translation of

mRNAs coding for components of translational apparatus

should be highly dependent on eIF4G, as well. Indeed,

polysome analysis of mRNAs after eIF4G knockdown showed

(Thoreen et al., 2012) that the translation of mRNAs highly

sensitive to mTOR inhibition (mainly 50 TOP and 50 TOP-like

mRNAs) is also sensitive to eIF4GI depletion. Thus, the

translation of mRNAs directly controlled by 4E-BPs depends

both on eIF4E (mostly eIF4E1) and eIF4G1, and, presumably,

on eIF4G3, the minor eIF4G variant with a domain organ-

ization identical to that of eIF4G1.

The mRNAs with a relaxed requirement for eIF4E butwith a strong dependence on eIF4G

It is well known that the binding of eIF4G to mRNA

dramatically enhances the binding of eIF4E to the 50 cap

(Haghighat & Sonenberg, 1997). In fact, some natural

mRNAs even possess special RNA structures within their 50

UTRs that facilitate recruitment of eIF4E�eIF4G complexes

(Wallace et al., 2010). It is easy to assume, therefore, that

different RNA sequences at the 50 termini of mRNAs would

have different affinity to eIF4G and thus could modulate the

requirement of the mRNA for eIF4E. Indeed, there are many

mRNAs whose translation is only marginally affected by

eIF4E depletion but strongly requires eIF4G. Many breast

cancer cells manifest an enhanced expression of eIF4GI and

concentration of this factor is critical for their survival after

irradiation therapy (Badura et al., 2012). eIF4G1 depletion

results in a significant drop in the survival of these tumor cells

(unlike knockdown of the less abundant eIF4G3) and in

growth arrest. Using microarray profiling of polysomal

mRNAs, a set of mRNAs was identified that were particularly

dependent on eIF4GI levels. They coded for survivin, XIAP,

HIF1a, GADD45a, p53, ATM, Chk1 and many other protein

factors involved in the DNA damage response (Badura et al.,

2012). eIF4E knockdown does not cause a strong effect on the

expression of these proteins (a maximum of 2-fold).

Conversely, eIF4G1 overexpression strongly improved sur-

vival, promoted assembly of the DNA Damage Response

complex (DDR-complex), suppressed apoptosis and overcame

autophagy. As shown in the same paper (Badura et al., 2012),

these properties were not accounted for by the presence of

IRES-elements within the corresponding 50 UTRs.

These data demonstrate that some mRNAs can do without

eIF4E, that is, they retain substantial translational activity

when it is deficient. A cellular mRNA that is independent of

the 50 cap and eIF4E, at least under stress conditions, but

nevertheless uses a 50 end-dependent mode of translation

initiation has been identified (Andreev et al., 2012). The

occurrence of mRNAs with such properties was additionally

supported by creating an artificial 50 UTR possessing such

properties (Terenin et al., 2013).

Other members of eIF4E and eIF4G families and theirpossible functions

The existence of homologues of both eIF4E and eIF4G

additionally expands the number of essential modifications of

the conventional mechanism of mRNA recruitment to 40S

ribosomes. Eukaryotic organisms have multiple eIF4E homo-

logues but the function of most of them is unknown (Joshi

et al., 2004, 2005; Rhoads, 2009). As a rule, one of them is

the most abundant and ubiquitous in every organism.

In mammalian cells this form is represented by eIF4E1.

As described above, it is mostly responsible for growth and

proliferation. It may be used by many, if not all mRNAs but

with different impacts on translation activity. Other forms are

presumably implicated in some specialized functions and

expressed at specific steps of development and differentiation.

For instance, Drosophila has eight eIF4E isoforms. One of

them, eIF4E2 (d4EHP), specifically interacts with bicoid

(Bcd) to suppress the caudal (cad) translation during devel-

opment. This translational suppression occurs via the Bcd

binding region (BBR) located in the cad 30 UTR.

Simultaneous interactions of eIF4E2 with the cap structure

and of Bcd with BBR renders cad mRNA translationally

inactive (Cho et al., 2005). Drosophila eIF4E3 (not to be

confused with mammalian eIF4E3) has been recently shown

to be essential for spermatogenesis (Hernandez & Vazquez-

Pianzola, 2005; Hernandez et al., 2012; Lasko, 2012).

DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 169

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Mammalian cells have three major forms of eIF4E:

eIF4E1, eIF4E2 (4EHP) and eIF4E3 (Joshi et al., 2004,

2005; Rhoads, 2009). Like in Drosophila, mammalian eIF4E2

does not interact with eIF4G and therefore is regarded as a

competitor for eIF4E1. Alone, it possesses a lower affinity for

the cap than eIF4E1 but we may not rule out that its

complexes with specific protein partners may modify this

affinity: a good example of this kind is the enhancement of

the cap-eIF4E interaction by eIF4G (Haghighat & Sonenberg,

1997; von Der Haar et al., 2000). Mammalian eIF4E2

interacts with the GIGYF2 protein and the zinc finger protein

598. This complex directs the inhibitory action of eIF4E2 to

specific mRNA targets to repress translation of a subset of

mRNAs during embryonic development (Morita et al., 2012).

The mechanism of repression is presumably similar to that

just described for eIF4E2 from Drosophila.

eIF4E3 forms a complex with eIF4G but does not bind

4E-BPs (Joshi et al., 2004). Its overexpression inhibits the

eIF4E1-cap interaction and therefore suppresses cellular

growth. The structural organization of its cap-binding surface

is strikingly distinct from that for eIF4E1 and 2 (Joshi et al.,

2004; Osborne et al., 2013). eIF4E3 is expressed only in some

tissues suggesting a role in translation of some specific

individual mRNAs rather than regulation of eIF4E1 – cap

recognition. It was found to be frequently deleted in oral

squamous cell carcinoma (Cha et al., 2011). We should note

again that the positive or inhibitory action of a particular

eIF4E homologue may be determined by protein partners with

which it interacts. For instance, eIF4E2 has recently been

shown to promote, rather than to inhibit, the translation

initiation of VEGF and other proteins under hypoxia (Uniacke

et al., 2012). To do this, eIF4E2 binds within the 30 UTRs of

corresponding mRNAs with assistance of auxiliary proteins,

one of which recognizing a specific nucleotide sequence. To

the best of our knowledge, this is thus far the only report on a

positive function of eIF4E2 in translation.

eIF4G also has several homologues (Figure 1A).

In different organisms, from yeast to mammals, there are

two homologous forms of this factor encoded by separate

genes, e.g. eIF4G1 (TIF4631) and eIF4G2 (TIF4632) in yeast

and eIF4G1 (eIF4GI) and eIF4G3 (eIF4GII) in vertebrates.

In addition, each has several variants arising from alternative

splicing, use of alternative promoters or variations in the

position of transcription starts (Byrd et al., 2005; Coldwell &

Morley, 2006; Coldwell et al., 2012). Interestingly,

Caenorhabditis elegans has a single gene for IFG-1, an

eIF4G homologue: the diversity of isoforms is maintained by

an alternative splicing and up to five isoforms have been

suggested to be synthesized. Importantly, the second major

isoform lacks a putative eIF4E-binding site and thus cannot be

purified via cap-binding chromatography (Contreras et al.,

2008). This raises the possibility that such truncated IFG-1

represents an ancient form of unusual eIF4G variant DAP5 in

mammalian cells (or eIF4G2, see below).

In general, various variants of eIF4G are able to function-

ally replace each other, at least partially. They are apparently

completely interchangeable in yeast (Clarkson et al., 2010).

In mammalian cells, the longest form of eIF4G3 is also

capable of substituting for eIF4G1 (Coldwell et al., 2012).

However, it seems very likely that specific combinations of

eIF4G and eIF4E forms do exist in higher eukaryotes.

Whether properties of eIF4F depend on which variants of

eIF4E and eIF4G form a complex is an intriguing question.

A Drosophila eIF4G variant, eIF4G-2, plays a critical role in

spermatocyte meiosis (Baker & Fuller, 2007; Franklin-

Dumont et al., 2007). It has been hypothesized that eIF4G-2

forms with eIF4E-3, a specific variant of eIF4F operating in

spermatocytes during this process (Hernandez et al., 2012).

Finally, the specialization of eIF4G isoforms can be achieved

through their specific phosphorylation (see Dobrikov et al.,

2014; Srivastava et al., 2012; and references therein).

Phosphorylation of eIF4E (Ser 209 in human eIF4E) by

MNK1/2 kinases in metazoans is one more way to diversify

the function of eIF4F. MNKs are recruited to eIF4E through

an interaction with the C-terminal part of eIF4G (Figure 1A).

It is poorly understood how this phosphorylation modifies

properties of eIF4E. On the one hand, the oncogenic potential

of unphosphorylatable mutant of eIF4E dramatically drops.

On the other hand, the phosphorylation was shown to reduce

its affinity to the cap. In addition to variable effects on general

translation, the phosphorylation of eIF4E was shown to

selectively affect translation of a subset of mRNAs, i.e. it may

serve as a translational switch between distinct classes of

mRNAs (for references, see Roux & Topisirovic, 2012).

eIF4F-independent ways of mRNA-binding toeukaryotic ribosomes

The existence of eIF4F-independent mode(s) of mRNA

recruitment to ribosomes has been strongly supported by an

identification of mRNAs that continue to be translated in

poliovirus infected cells (Macejak & Sarnow, 1991). The

poliovirus infection is known to produce the viral protease 2A

that rapidly cleaves eIF4G1 between amino acid 681 and 682

(and with a much slower kinetics also eIF4G3) (Figure 1A).

The cleavage disconnects the eIF4E binding site of eIF4G

from those for eIF4A and eIF3, thereby abolishing the

recruitment of mRNA to the 40S subunit by a cap-dependent

way. It has been thought for a long time that the continued

translation of some mRNAs in poliovirus-infected cells is

indicative of an IRES-driven mode (Figure 2B) of translation

initiation. More recent data strongly suggest that 50 UTRs of

some mammalian mRNAs can do without the cap (and

therefore without eIF4E) even if they have no IRES (Andreev

et al., 2009, 2012; Terenin et al., 2013).

Mammalian eIF4G2 (other names: Death Associated

Protein 5 (DAP5), Novel APOBEC Target1 (NAT1) or p97;

not to be confused with eIF4GII, or eIF4G3) deserves special

attention (Figure 1A). This mysterious version of eIF4G was

discovered simultaneously in 1997 by four separate labora-

tories even though each group worked in quite different fields

(Imataka et al., 1997; Levy-Strumpf et al., 1997; Shaughnessy

et al., 1997; Yamanaka et al., 1997). eIF4G2 (MW 97 kDa)

appears to be an N-terminal truncated form of eIF4G which

lacks binding sites for eIF4E and PABP. At least in higher

eukaryotes its coding sequence invariably starts with a non-

AUG codon (always GUG in vertebrates) (Takahashi et al.,

2005). eIF4G2 demonstrates a non-continuous distribution

among Metazoans, being present in all Chordata and some

invertebrates (insects, sea urchins, mollusks and some types

170 I. N. Shatsky et al. Crit Rev Biochem Mol Biol, 2014; 49(2): 164–177

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of worms – but unexpectedly absent from C. elegans where it

may be replaced by a truncated variant of eIF4G, see above).

Such a complex distribution suggests a role for eIF4G2 in

some higher functions associated with multicellularity. Since

it cannot associate with eIF4E, eIF4G2 has long been

regarded as a negative regulator of cap-dependent translation

which is transformed into a positive regulator after cleavage

with Caspase-3 into p86 under apoptotic conditions (Marash

& Kimchi, 2005). It is believed to serve some cellular mRNAs

that harbor IRES-elements (Figure 2B) operating under stress

conditions. However, this hypothesis still requires experi-

mental support (see next section about cellular IRESs).

Surprisingly, apart from apoptosis (Marash & Kimchi, 2005;

Ozpolat et al., 2008), eIF4G2 has been implicated in the

opposite responses, e.g. in cell survival during mitosis and

differentiation (Liberman et al., 2009; Marash et al., 2008).

Null mutation of eIF4G2 in mice leads to an embryonic

lethality since the factor is essential for gastrulation. The

expression of retinoic acid-responsive genes, such as the cell-

cycle inhibitor p21 (WAF1), is also selectively impaired in

eIF4G2 (�/�) cells (Yamanaka et al., 2000). Knockdown of

eIF4G2 expression by siRNAs suppresses all-trans-retinoic

acid-induced granulocytic differentiation (Ozpolat et al.,

2008).

Overall, the data obtained in the first 10 years after the

discovery of eIF4G2 gave the impression that this factor is

implicated in translation of a rather small subset of mRNAs

involved in highly specialized functions. However, later

reports, including transcriptome-wide studies, suggest that

eIF4G2 governs the translation of a larger set of mRNAs and

may be required by cells growing under normal conditions

(Lee & McCormick, 2006; Nousch et al., 2007; Ramirez-

Valle et al., 2008). eIF4G2 was found to be associated with

the polysome fraction in actively proliferative cells (Nousch

et al., 2007). Remarkably, depletion on eIF4G1 alone by

siRNAs resulted in only 20–30% inhibition of total protein

synthesis, whereas simultaneous depletion of both eIF4G1

and eIF4G2 leads to 60% inhibition and cessation of cell

division. Moreover, overexpression of eIF4G2 does not

compensate the eIF4G1 depletion indicating that these two

factors direct translation of distinct sets of mRNAs (Ramirez-

Valle et al., 2008). Like all other members of the eIF4G

family, eIF4G2 contains the MIF4G domain that binds

helicase eIF4A albeit with a lower affinity than the respective

domain from eIF4G1 (Virgili et al., 2013). The mechanism of

functioning and partners of eIF4G2 are not known. It is

ubiquitous and relatively abundant in both cultured cells and

normal tissues (Geiger et al., 2013; Schwanhausser et al.,

2011) supporting the view that this factor is a translational

activator of many mRNAs even under normal conditions.

The eIF4G2 is not the only unusual homologue of eIF4G1/

3 with the MIF4G domain. In recent years, a number of other

MIF4G domain – containing proteins have been further

investigated (Figure 1B). They seem to escort eukaryotic

PolII-transcripts through all the steps of RNA metabolism:

starting from transcription and splicing (CBP80, NOM1/

SGD1, CWC22), through nuclear export and mRNA quality

control (all the above plus GLE1 and UPF2) to translation in

the cytoplasm (CBP80, GLE1, PAIP1, CTIF and SLIP1) and

to mRNA decay (NOT1). However, not all of them may be

regarded as factors involved in mRNA recruitment to

ribosomes, i.e. the process discussed in this review. Other

MIF4G proteins may be implicated in later steps of translation

initiation or its regulation (see, for example, Bolger & Wente,

2011). Complex of CBP80 and nuclear cap-binding protein

CBC20 is thought to be involved in the pioneer round of

translation of mRNAs (Maquat et al., 2010) exported out of

the nucleus. The mRNA recruitment to ribosomes with this

factor is assisted by CTIF (CBP80/20-dependent Translation

Initiation Factor) and eIF3g (Choe et al., 2012; Kim et al.,

2009). However, it is quite possible that CBP80/20 complex is

also involved in translation of a specific set of mRNAs, e.g.

the mRNAs activated under stress conditions (Sharma et al.,

2012). Its yeast functional analog cbc1 plays an active role in

translation: it is necessary for rapid reprograming of trans-

lation after hyper-osmotic shock (Garre et al., 2012). Paip1

stabilizes the interaction between eIF4G and PABP by

binding to eIF3, which in turn binds to eIF4G and the

40S ribosome (Martineau et al., 2008). The factor SLIP1

is needed for translation initiation on some replication-

dependent histon mRNAs which are not polyadenylated and

instead have a specific stem-loop structure at the 30 end

(Cakmakci et al., 2008; Neusiedler et al., 2012; von Moeller

et al., 2013).

Hernandez et al. have recently reported the discovery of

Mextli (Mxt), a novel eIF4E-binding protein in Drosophila

that promotes translation (Hernandez et al., 2013). It contains

(Figure 1B) a MIF4G domain, an hnRNP K homology

RNA-binding domain and a consensus eIF4E-binding site.

Strikingly, its eIF4E binding site is located at the C-terminus

of the factor. Mxt is expressed at high levels in ovarian

germline stem cells and early-stage cystocytes and forms a

complex with both eIF4E1 and eIF4E3. Thus, Mxt is a novel

type of scaffolding protein that binds RNA, eIF3 and eIF4Es.

Interestingly, Mxt interacts with a DEAH-box helicase

CG3225 (ortholog of mammalian DHX35) instead of

‘‘canonical’’ eIF4A. Although Mxt was not found in mam-

malian cells, its discovery is a very important event since it

hints at the existence of other non-canonical eIF4G analogs in

eukaryotic cells.

An even more intriguing question concerns the possible

participation of ‘‘alternative’’ cap-binding complexes in

translation initiation under certain conditions. For example,

a proteomic study of m7G-associated proteins from quiescent

plant cells revealed an unexpected set of polypeptides where

eIF4A was completely absent but several other DEAD-box

proteins (plant-specific or homologous to mammalian

DDX3X, DDX3Y or DDX16) appeared, together with nuclear

cap-binding protein CBP20 (Bush et al., 2009). In another

study, translation induction of a specific mRNA set encoding

enzymes of the pentose phosphate pathway was detected after

glucose depletion conditions that induced eIF4A loss from

eIF4F (Castelli et al., 2011).

The possibility of eIF4F- and eIF4G-independent modes of

translation initiation in eukaryotic cells is indirectly supported

by experiments with eIF4G depletion. As mentioned above,

its knock-down in mammalian cells resulted in a surprisingly

small effect. It strongly affected only 20% of cellular mRNAs

(Ramirez-Valle et al., 2008). Although the eIF4G depletion in

yeast cells was eventually lethal, it had a small effect on the

DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 171

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translational potential of most mRNAs (Park et al., 2011b).

Depleting eIF4G just narrowed the range of translational

efficiencies transcriptome-wide: the yeast mRNAs with better

than average efficiency were translated relatively worse,

whereas the mRNAs with lower than average efficiency were

translated relatively better. Importantly, the mRNAs most

dependent on eIF4G showed an average 50 UTR length at or

below the mean for all yeast genes. The authors even

concluded that eIF4G (eIF4GI + eIF4GII) is not essential for

translation of most mRNAs but enhances the differentiation of

translational efficiencies transcriptome-wide (Park et al.,

2011b).

The concept of cellular IRES-elements twodecades later

Many researchers, in particular those who are not specialized

in translation, believe that the cap-independent mode of

translation initiation is synonymous to IRES-driven initiation.

The data discussed in the previous section of this review

strongly contradict this view. Nevertheless, the concept of

cellular IRESs as a principal cap-independent mechanism of

translation initiation in eukaryotes is still dominant since it

explains the deviations from the standard mechanism in a

plausible, simple and hence a very attractive way.

Virus IRES-elements are no longer a scientific hypothesis.

Their existence is a firmly established fact. Some IRESs from

viruses with a positive RNA genome are characterized in

detail (for recent review see, Jackson, 2013) though several

important gaps still remain. Based on their structures, factor

requirements and modes of function, the virus IRESs are even

amenable to some classification. In contrast, in spite of the

22-years long history (Macejak & Sarnow, 1991) of cellular

IRESs, we do not know yet what they look like, how they

work and even whether they exist. The existence of none of

them has been proven with the use of a set of rigorous control

experiments. Most of them have been proposed on the basis of

cell transfection experiments with dicistronic DNA construc-

tions, i.e. by an approach extremely prone to unavoidable

artifacts (reviewed by Shatsky et al., 2010). Recent work by

Lemp et al. (2012) who detected cryptic transcripts from an

ubiquitous plasmid origin of replication used in an over-

whelming majority of reporter plasmids has finally high-

lighted the severe short-comings of using DNA-based

constructs for IRES analysis. A set of strict criteria that

should be met and a set of control experiments that should be

performed before researchers confirm the existence of a

cellular IRES are presented by Andreev et al. (2009) and

Jackson (2013). One of the most important criterion, the ratio

of absolute translation efficiencies of a 50 UTR in the capped

monocystronic context versus dicistronic position, is routinely

absent from published reports. As a result, readers cannot

judge whether the authors deal with a physiologically relevant

IRES or with a background activity.

Another widely held opinion, which is used to support the

concept of cellular IRESs, is that the scanning 50 end-

dependent mechanism cannot operate in cells with an

inactivated eIF4E or cleaved eIF4G1 can also be challenged

(Andreev et al., 2012; and see the text above). Finally, an

mRNA with a low cap-dependence that still requires the 50

end has been recently demonstrated by our lab to be highly

translationally active. To this end, we created a model mRNA

that had such properties: its translation depended very little on

the cap both in vitro and in transfected cells and occurred via

a 50 end-dependent scanning mechanism (Terenin et al.,

2013). Additionally, this report indicated that the presence

within the 50 UTR of a high affinity binding site for a key

translation initiation component (factor) was necessary but

not sufficient for internal initiation. Indeed, the well-

characterized virus IRESs appear to harbor, along with such

an affinity site, additional specific domains that promote

placing the initiation region of the mRNA into the

mRNA-binding channel of the ribosome. The best studied

example is domain II of HCV IRES (Filbin & Kieft, 2011;

Malygin et al., 2013). Otherwise, if such an accommodation

‘‘device’’ is lacking, the only point at which the process of

mRNA ‘‘threading’’ into the 40S subunit channel can be

initiated is the very 50 end of the mRNA (Figure 2). The

requirement for a free 50 terminus is not determined

exclusively by the cap and eIF4E. Even more it is dictated

by a great preference that the scanning machinery has for an

unpaired mRNA end to start unwinding stem-loops within 50

UTRs of mRNAs (Rajagopal et al., 2012 and references cited

there in). The only exclusion from this general rule may be

A- or U-rich nucleotide tracts: their lower base-pairing

potential may enable them to spontaneously occupy the

mRNA binding groove of the 40S ribosome without assist-

ance of the RNA-unwinding machinery (Gilbert et al., 2007;

Terenin et al., 2005).

Finally, compelling evidence that the initiation from a free

50 end is only partially determined by recognition of the

cap by eIF4E is offered by RNAs from some plant viruses.

The mRNAs of viruses from the Potyviridae, Comoviridae,

Tombusviridae and Luteoviridae families and the

Sobemovirus and Umbravirus genera lack the m7GpppN-

cap structure. These viruses employ special elements termed

Cap Independent Translation Enhancers (CITE) (Figures 2C

and D) (reviewed by Miller et al., 2007). The majority of

CITEs are located in the 30 UTRs (30 CITEs). They are

thought to recruit components of the translational apparatus

(e.g. eIF4E or eIF4G) and then deliver them to the 50 end of

mRNA through long distance base pairing between 50 and 30

UTRs (Nicholson et al., 2010; Stupina et al., 2008; Treder

et al., 2008). The CITE’s can also function when placed

within 50 UTRs of mRNAs (Guo et al., 2000) (Figure 2C).

The initiation on these plant virus RNAs starts at the 50 end

and employs a scanning mechanism. We have suggested that a

similar mechanism may also be used by mRNAs from animal

cells (Shatsky et al., 2010) and demonstrated its feasibility

(Terenin et al., 2013).

Thus, there may be two quite different cap-independent

ways of mRNA recruitment onto the 40S ribosomal subunit in

mammalian cells, an IRES-driven mode and a 50 CITE-based

mechanism (Figure 2B and C). They are easy to distinguish:

the latter mode should manifest a very low translation activity

when the corresponding nucleotide sequence is placed

into the intercistronic position of a dicistronic mRNA in

comparison with that in a natural capped monocistronic

context. For a true IRES-element, these activities should be

quite similar. This test (capped monocistronic versus

172 I. N. Shatsky et al. Crit Rev Biochem Mol Biol, 2014; 49(2): 164–177

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Figure 3. Speculative modes of mRNA recruitment to ribosomes used by distinct classes of cellular mRNAs. (A) The mRNAs that are highlydependent on eIF4E availability: blocking of eIF4E by dephosphorylated 4E-BPs results in dissociation of eIF4G + eIF4A from such mRNAs and theirtranslational inactivation. (B) The mRNAs which are less dependent on the eIF4E – m7G cap interaction and therefore less sensitive to mTORinhibition. These mRNAs may have a higher affinity of eIF4G + eIF4A to their 50 UTRs (may have CITEs) and remain active in the translationinitiation even under complete inhibition of eIF4E. (C) Specialized combinations of eIF4E and eIF4G. Specific variants of these factors are denoted aseIF4Ex and eIF4Gx. The corresponding forms of eIF4F may bind to particular mRNAs and activate them for translation during development anddifferentiation. Some of these combinations may be refractory to standard 4E-BPs. (D) The mRNAs that use eIF4G2 or eIF4G-like factors rather thaneIF4F. These factors may recognize the corresponding 50 UTRs with the help of auxiliary mRNA binding proteins (denoted with ‘‘Y’’). Such mRNAsare not necessarily cap-independent: they may use non-canonical cap-binding molecules (denoted as ‘‘X’’) which communicate with eIF4G-like factorsthrough specific mRNA-binding proteins. Alternatively, such mRNAs may employ IRES- or CITE-based mechanisms. All the modes of mRNArecruitment which are illustrated in this figure require a free 50 terminus and involve scanning from the 50 end. The IRES-driven translation initiation isdepicted in Figure 2(B).

DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 173

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intercistronic) should be performed both under normal and

stress conditions, to check whether or not the putative IRES is

induced by stress.

Concluding remarks and hypotheses

A growing body of evidence hints at the existence of

significant modifications in the standard mechanism of

mRNA recruitment to eukaryotic ribosomes. They may

differ in the set of recruitment factors or their concentrations.

In addition, subsets of mRNAs may employ, at least under

some specific conditions, mechanisms that do not involve

eIF4E or even eIF4F. We do not exclude that IRES-elements

may also be implicated in the translation initiation of cellular

mRNAs. We only stress that all putative IRESs should be

verified and carefully characterized using stringent criteria to

show that internal ribosome binding activities do have

reasonable and physiologically justified values. These

values should be at least comparable with those of the 50

end-dependent initiation for the same 50 UTRs. On the

basis of information available to date, we can propose

several speculative modes of mRNA recruitment onto 40S

ribosomes that are used by distinct classes of eukaryotic

mRNAs:

Class 1. Highly eIF4E- and eIF4G-dependent mRNAs

(Figure 3A): The number of such mRNAs is relatively small

but they make up a substantial portion of the total amount of

mRNA within polysomes of proliferating cells. Their trans-

lation rapidly responds to changes in nutrient availability and

growth factors. They are strongly and directly controlled by

mTORC1, mostly via phosphorylation of 4E-BPs and are

represented by mRNAs responsible for growth and prolifer-

ation. Interestingly, restriction of their translation extends life

span (Harrison et al., 2009; Syntichaki et al., 2007; Zid et al.,

2009). They are overwhelmingly represented by mRNAs that

encode components of translational apparatus (ribosomal

proteins, some initiation factors, elongation factors eEF1 and

eEF2, PABP, etc.) as well as some factors participating in cell

cycle and epithelial-mesenchymal transition. All 50 TOP

mRNAs belong to this class. Their translation initiation is

strongly dependent on the concentration of free eIF4E, but

also dependent on eIF4G1/3. Importantly, eIF4G alone is not

able to support their efficient recruitment to 40S ribosomes,

presumably, because of a low affinity to the corresponding

UTRs. That is why disruption of the interaction of eIF4E with

eIF4G upon 4E-BPs activation results in a rapid dissociation

of eIF4G and eIF4A from such mRNAs. This process may be

additionally promoted by 50 TOP-binding proteins, TIA-1 and

TIAR (Damgaard & Lykke-Andersen, 2011).

Class 2. eIF4G-controlled mRNAs (Figure 3B): mRNAs of

this class may contain within their 50 UTRs sites with a high

affinity to eIF4G. Such mRNAs require less eIF4E or can

even do without this factor (e.g. when they harbor CITEs),

especially in non-dividing cells where the competition for

ribosomes with mRNAs of class 1 is low or even absent. It is

not excluded that a more stable and specific binding of eIF4G

to a 50 UTR may produce conformational changes in this

factor which in turn enable a more stable binding of eIF4E.

In this case, these mRNAs might successfully compete with

4E-BPs for eIF4E binding. It is worth mentioning that the

translation of the mRNAs which code for all eIF4G variants is

resistant to mTOR inhibition (4E-BPs activation) (Thoreen

et al., 2012).

Class 3. mRNAs controlled by specific variants of eIF4E,

eIF4G and their combinations (Figure 3C): As noted above,

the use of alternative forms of eIF4E and eIF4G during

mitosis and development is well documented, especially in

Drosophila. It is logical to predict the existence of their

specific combinations operating with particular mRNAs.

However, this issue has not been explored and we do not

know how these alternative forms and combinations are

selected by specific mRNAs. It may well be that this selection

is assisted by other auxiliary factors or mRNA-binding

proteins.

Class 4. mRNAs that do not use eIF4F (Figure 3D): They

may utilize a truncated form of eIF4G1, eIF4G2 (DAP5) or

other functional analogs of eIF4G1 (eIF4G-like proteins). The

mRNAs controlled by such factors are not necessary cap-

independent. They may use a cap-binding protein that

communicates with an eIF4G-like protein via a specific

mRNA-binding protein. Alternatively, DAP5 or similar

proteins may be a critical factor that governs internal

initiation on some cellular IRESs.

Finally, we have no idea about the factor requirement for

ribosome recycling through preformed polysomes. It may

well be that polysomes assembled on some mRNAs no longer

need eIF4E, eIF4G or even eIF4A for new rounds of

translation. This could be an alternative explanation why

eIF4G depletion produced a relatively minor effect on

eukaryotic translation. Some fragmentary data on insensitiv-

ity of preformed polysomes to a cap-analog (Amrani et al.,

2008) or non-hydrolysable derivative of ATP (Kopeina et al.,

2008) should encourage further experiments in this direction.

Certainly, there may be many more specific mechanisms

that determine translation initiation on eukaryotic mRNA.

This may be an exciting direction for further studies

of eukaryotic translation and its regulation. In addition,

knowledge of these mechanisms would be useful if we want

to design drugs targeting the translation of specific classes

of mRNAs in particular cells. To succeed in this tremendous

and very ambitious task, we should intensify not only

transcriptome-wide studies of translation but also focus

special attention on developing advanced biochemical tech-

niques and sophisticated in vitro approaches to study the

underlying mechanisms in detail. Otherwise, our knowledge

will remain just a myriad of effects, observations and their

arbitrary interpretations. Development of such techniques

certainly lags behind genome- and transcriptome-wide

studies.

Acknowledgements

We apologize to our colleagues for not being able to discuss

other important contributions due to space constraints. The

authors are very grateful to Gary Loughran (Cork Institute,

Ireland) for critical reading of the manuscript and valuable

suggestions.

Declaration of interest

The authors report no declaration of interest.

174 I. N. Shatsky et al. Crit Rev Biochem Mol Biol, 2014; 49(2): 164–177

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We acknowledge financial support from the Russian

Foundation of Basic Researches to I.N.S. (grant N 11-04-

01010) and S.E.D. (grant N 12-04-33196).

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DOI: 10.3109/10409238.2014.887051 Transcriptome-wide studies of mRNA binding to ribosomes 177

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