The RNPs of eukaryotic translation control
Protein synthesis is the essential cellular process
of translating the genetic code into the major
structural and functional biomolecule of the cell:
the protein. Eukaryotic translation is a dynamic
molecular process choreographed by translation
ribonucleoprotein (trRNP) complexes that assemble
upon a messenger RNA (mRNA) and regulate its
interaction with the bimolecular catalyst of this
event, the ribosome. From their transcription, mRNAs
assemble within dynamic RNA-protein structures
that regulate their stability, processing, localization,
and ultimately their translation. Ribonucleoprotein
(RNP) complex is a term used to broadly define
these RNA-protein assemblies. trRNP complexes
specifically relate to dynamic mRNA-protein
structures that coordinate the translation process.
trRNPs are at the core of eukaryotic translation
control. They are endowed with the ability to regulate
the localization, conformation, and activation of
mRNAs, all features that regulate engagement with
the ribosome and generation of protein product.
This review provides a comprehensive analysis
of the trRNPs of eukaryotic translation control.
Mechanisms known for regulating eukaryotic trRNP
activity will be discussed with reference to their
significance in cell biology. The importance of
distinct trRNPs in selective translation control
will be highlighted with a specific focus on the
DExH/D-box RNA helicase trRNPs and those of
unique RNA binding proteins. The outcome is an
enhanced understanding and appreciation for the
role of RNP biology in the regulation of protein
KEYWORDS: protein synthesis, eukaryotic
translation control, translation RNP (trRNP), CBC
trRNP, eIF4E trRNP, DExH/D-box RNA helicases
Eukaryotic translation occurs in three mechanistic
stages: initiation, elongation, and termination. Each
stage is coordinated by a set of translation factors,
which are RNA binding and/or scaffolding proteins
that assemble upon an mRNA and regulate its
engagement with the ribosome. These RNA-protein
complexes give rise to the trRNPs of eukaryotic
translation control. The integration of distinct
RNA binding proteins, such as DExH/D-box
RNA helicases, into this process creates selective
trRNPs important for targeted protein synthesis.
Initiation is the rate-limiting step of protein
synthesis with numerous regulatory mechanisms
identified to control its activation and efficiency.
The defining principle of eukaryotic translation
initiation is a cap-dependent scanning mechanism
whereby the ribosome binds to the 5' terminus of
an mRNA and proceeds along the transcript,
inspecting base-by-base, for an appropriate start
codon to initiate polypeptide synthesis . This
process requires at least nine initiation factors
(eIFs) and begins with the activation of an mRNA
and its association with the 43S pre-initiation
ribosome complex  (Figure 1).
Department of Veterinary Biosciences, Center for Retrovirus Research, Center for RNA Biology,
Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
Sarah Fritz and Kathleen Boris-Lawrie*,#
*Corresponding author: firstname.lastname@example.org
#Current address: Department Veterinary & Biomedical
Sciences, University of Minnesota, Saint Paul,
MN 55108, USA.
Vol. 10, 2015
106 Sarah Fritz & Kathleen Boris-Lawrie
RNA binding proteins target select mRNA templates for translation 107
[1-3] (Figure 1). This places the 40S ribosomal
subunit and associated initiator methionyl-tRNA
in a conformation that is conducive for movement
along an mRNA and for base-by-base inspection
[1-3]. Appropriate anticodon-codon base pairing
between the initiator methionyl-tRNA and a start
AUG triggers a conformational rearrangement in
the 43S pre-initiation ribosome complex that causes
the mRNA entry channel latch to close, stopping
the scanning process [1-3] (Figure 1). This repositions
the initiator methionyl-tRNA and 40S ribosomal
subunit in a favorable context for joining of the
large (60S) ribosomal subunit and formation of
elongation-competent 80S ribosomes [1-3].
Interactions between mRNA sequence, eIF2 and
the 18S rRNA facilitate favorable anticodon-
codon base pairing that halts ribosome scanning
[1-3]. Contacts between eIF2 and the 18S rRNA
with distinct purine nucleotides at positions -3 and
+1 relative to the adenine nucleotide within an
identified AUG codon defines an optimal sequence
context to end ribosome scanning and initiate
polypeptide synthesis [1-3]. These associations
induce conformational changes within the 43S
pre-initiation ribosome complex that tighten the
interaction of eIF1A with the 40S ribosomal
subunit, displacing eIF1 and resulting in a ‘closed’
conformation of the mRNA entry channel latch
with the initiator methionyl-tRNA oriented in an
inward, locked position primed for subsequent
peptide bond formation [1-3]. Ejection of eIF1
releases an inhibitory block on both ribosome
conformation and eIF5-regulated eIF2 GTPase
Eukaryotic mRNAs are defined by a 5' 7-methyl
guanosine cap, which is bound co-transcriptionally
and throughout the lifespan of an mRNA by a cap-
binding protein. The cap-binding protein, CBP80/20
or eIF4E, and its associated scaffolding factor,
CTIF and/or eIF4G, generate a trRNP that primes
an mRNA for translation (discussed further in
section ‘The CBC and eIF4E trRNPs’). This
activation encompasses circularization of the mRNA
via an interaction between the 5' scaffolding factor
and the 3'-bound poly-A binding protein (PABP)
to result in recruitment of the 43S pre-initiation
ribosome complex to the 5' terminus of a transcript
[2, 3] (Figure 1). The 43S pre-initiation ribosome
complex consists of the small (40S) ribosomal
subunit, initiator methionyl-tRNA, and five core
initiation factors (eIF1, 1A, 2, 3 and 5), each with
a distinct role in the recruitment and scanning
process . Interactions between eIF3 of the 43S
pre-initiation ribosome complex and the 5'
mRNA-bound scaffolding factor, CTIF and/or eIF4G,
drive the association of the 43S pre-initiation
ribosome complex with the activated mRNA [2-4]
Once associated with a target mRNA, the 43S pre-
initiation ribosome complex begins the scanning
process in search of an appropriate start codon to
initiate polypeptide synthesis. eIFs 1 and 1A are
critical for facilitating this process. The positioning
of eIFs 1 and 1A within the 43S pre-initiation
ribosome complex confers structural arrangements
that stabilize an ‘open’ conformation of the mRNA
entry channel latch within the 40S ribosomal subunit
Legend to Figure 1. Schematic of canonical eukaryotic translation initiation. Canonical eukaryotic translation
initiation is characterized by a cap-dependent scanning mechanism whereby the ribosome binds to the 5' terminus of
an mRNA and proceeds along the transcript, inspecting base-by-base, for an appropriate start codon to initiate
polypeptide synthesis. This process begins with the activation of an mRNA and its association with the 43S pre-
initiation ribosome complex (43S PIC). Interactions among the 5' associated scaffolding factor, CTIF or eIF4G, and
the 3'-bound poly-A-binding protein (PABP) circularizes an mRNA to generate a translation ribonucleoprotein
(trRNP) complex that engages with the 43S PIC. This occurs via stimulated interactions between the scaffolding
factor and eIF3. 5' to 3' scanning follows, as the 43S PIC searches for an optimal start codon (AUG) to initiate
polypeptide synthesis. Directed movement is facilitated by initiation factors eIF1 and eIF1A. Their positioning
within the 43S PIC stabilizes an ‘open’ conformation of the mRNA entry channel latch and orients the initiator
methionyl-tRNA in an outward position that is conducive for scanning. Start codon recognition occurs when an
AUG is reached within an optimal sequence context and appropriate anticodon-codon base pairing occurs. This
induces a conformational rearrangement in the 43S PIC that causes the mRNA entry channel latch to close and the
scanning process to stop. Repositioning of eIF1A stimulates eIF1 release and rotation of the initiator methionyl-
tRNA into an inward position that is favorable for 60S ribosomal subunit joining. Joining of the large (60S)
ribosomal subunit is facilitated by eIF5B and results in the formation of an elongation-competent 80S ribosome
primed for polypeptide synthesis.
108 Sarah Fritz & Kathleen Boris-Lawrie
a ‘loop-out’ effect of the 5' untranslated region
whereby the scanned mRNA bulges as the ribosome
continues forward . This outcome would
effectively allow only one scanning ribosome at a
time . Alternatively, the connections between
the 43S pre-initiation ribosome complex and the
5' cap could be broken, allowing several ribosomes
to translocate a 5' terminus simultaneously .
Advances in single molecule imaging technology
and their application to understanding translation
control will provide clarification on the fate of
the initial recruitment connections during the
The final step in the initiation stage of translation
is joining of the large (60S) ribosomal subunit
with the small (40S) ribosomal subunit at the start
codon to generate an elongation-competent 80S
ribosome (Figure 1). The translation initiation factor
eIF5B mediates this effect. The presence of eIF5B
and the 60S ribosomal subunit stimulates complete
release of eIF2-GDP and eIF5 from the stopped
43S pre-initiation ribosome complex at the start
AUG . This dissociation allows for the interaction
of eIF5B in a GTP-bound form with eIF1A, which
remains temporarily associated with the 40S
ribosomal subunit . Binding of the 60S ribosomal
subunit then follows, stimulating the GTPase
activity of eIF5B . The hydrolysis of GTP to
GDP weakens the affinity of eIF5B for the ribosome,
resulting in its release . eIF1A subsequently
dissociates to leave an 80S ribosome primed for
Several mechanistic aspects remain to be defined
for this final stage of translation initiation. First, it
is unknown how eIF5B and the 60S ribosome are
recruited to a 43S pre-initiation ribosome complex
. Is eIF5B a component of this multifactor complex
that then recruits the 60S ribosomal subunit when
in an optimal conformation? Alternatively, does
the recruitment of eIF5B to the 43S pre-initiation
ribosome complex draw in the 60S ribosome or
simply increase the likelihood of interactions
between the small and large ribosomal subunits?
Second, it is unclear as to what the molecular
significance of the eIF5B-eIF1A interaction is in
facilitating 60S ribosomal subunit joining .
Does the interaction of eIF5B with eIF1A stimulate
conformational rearrangements in the 40S ribosomal
subunit that favor appropriate 60S binding? Or
activity that results in phosphate release, initiation
factor dissociation and 60S ribosomal subunit
Outstanding questions remain in regard to the
mechanism(s) underlying net 5' to 3' movement of
the 43S pre-initiation ribosome complex as well
as the fate of the initial recruitment connections
during the scanning process. Studies of dicistronic
polypeptide production from eukaryotic viral
transcripts indicate an oscillating 43S pre-
initiation ribosome complex with both forward
and reverse movements . This fluctuating
motion is critical to sequence surveillance and
selection of an AUG for initiation of polypeptide
synthesis . Structural analyses support these
molecular findings by presenting the ribosome as
a ‘processive Brownian motor’ . This model
proposes that movement of the ribosome along
a transcript is directed by interactions with the
nearby environment, such as collisions with mRNA
secondary structure or associations with protein
cofactors. The conformational changes that result
permit both forward and reverse movements of
the ribosome that are intricate to the selection
of an AUG for initiation of polypeptide synthesis.
A sum of these localized fluctuations results in a
net outcome of observed 5' to 3' directionality in
The initial attachment of a 43S pre-initiation
ribosome complex to an activated mRNA requires
interactions between eIF3 of the 43S pre-initiation
ribosome complex and the 5'-bound scaffolding
factor eIF4G or CTIF [2-4]. eIF4G also directly
associates with the cap-binding protein eIF4E,
regulating the stable association of eIF4E with
the 5' cap . The additional RNA binding activity
of eIF4G facilitates this effect . Furthermore,
eIF4G directly associates with the 3' poly-A
binding protein (PABP) to generate a circularized
mRNA that is effective for 43S pre-initiation
ribosome complex recruitment [2, 7]. Collectively,
these eIF4G-driven associations link the 5' cap of
a target mRNA with the 43S pre-initiation ribosome
complex and are intricate to its recruitment and
scanning activity. However, it remains to be
determined whether these associations persist
during ribosome scanning of the 5' terminus. If a
connection remains between the 5' cap and 43S
pre-initiation complex then the outcome supports
RNA binding proteins target select mRNA templates for translation 109
of a core helicase domain within their protein
structure that harbors nine conserved motifs
involved in RNA binding, nucleotide triphosphate
association, and its hydrolysis [8-11]. The
DExH/D-box RNA helicases are identified by a
distinct Asp-Glu-x-His/Asp (DExH/D) core within
motif II, which lies within the center of the
helicase domain and is critical for association with
the β- and γ-phosphates of a nucleotide triphosphate
[8-11]. Although conservation of sequence and
function are critical for the classification of
DExH/D-box RNA helicases, variations in the
defining helicase core along with differences in
flanking domains result in distinct trRNP association
and function for each DExH/D-box RNA helicase
eIF4A (DDX2) is the DEAD-box RNA helicase
most understood for regulating translation of
eukaryotic mRNAs . Functioning as an ATP-
dependent RNA binding protein, eIF4A separates
localized duplexed strands by coupling ATP-driven
enzymatic changes to RNA unwinding .
does an eIF5B-eIF1A association engage more in
active recruitment? Ongoing structural studies are
aimed at providing greater mechanistic understanding
into the significance of eIF5B and eIF1A in the
generation of elongation-competent 80S ribosomes.
DExH/D-box RNA helicase trRNPs
Intricate to mRNA activation and ribosome
recruitment are DExH/D-box RNA helicases.
DExH/D-box RNA helicases are both RNA
binding proteins and enzymatic catalysts that
couple the free energy of nucleotide triphosphate
hydrolysis to RNA unwinding and/or RNP
remodeling [8-11]. The outcome is dynamic
structural rearrangements within the trRNP that
regulate placement and then movement of the 43S
pre-initiation ribosome complex along the 5'
terminus of an mRNA .
Over fifty DExH/D-box RNA helicases have been
identified with critical roles in post-transcriptional
gene control. They are recognized by the presence
Table 1. Known features of select DExH/D-box RNA helicase trRNPs.
DHX29 RNA helicase A
1. Cognate cis-
G-quadruplex Stable stem
and G-C rich
and G-C rich
CBC trRNP + + - To be determined
eIF4E trRNP + - + + To be determined
To be determined
translation Enhancer of type
I and II Enhancer of
type III and
110 Sarah Fritz & Kathleen Boris-Lawrie
reads at particular positions along the target
mRNAs . Distinct patterns that emerge from
these results inform subsequent analyses that
allow for identification of molecular signatures
defining a particular translation landscape.
In the case of eIF4A, treatment of cells with
silvestrol, a direct inhibitor of eIF4A helicase
activity , revealed that eIF4A-sensitive transcripts
are specifically defined by a 12 or 9-nucleotide
(CGG)4 motif within their 5' termini . This
(CGG)4 motif forms a stable G-quadruplex secondary
structure, sensitizing mRNAs to eIF4A helicase
activity and identifying the eIF4A translational
landscape . This eIF4A translational landscape
encompasses many prominent transcriptional
regulators, including several super-enhancers ,
providing a molecular basis for the vast expression
reprogramming observed upon mutation of eIF4A,
even given its newly identified specificity in
translation control. Thus, the significance of eIF4A
in 43S pre-initiation ribosome complex recruitment
and scanning is specific for a subset of mRNAs
harboring its cognate cis-acting RNA element, the
The repertoire of cis-acting structural elements
found within the 5' termini of eukaryotic mRNAs
is diverse, and extends beyond G-quadruplex
structures to include various stem and internal
loops, bulges, junctions and pseudoknots. The
DExH/D-box RNA helicases eIF4AIII (DDX48),
DDX3, DHX29, and RNA helicase A (RHA/DHX9)
are intricate to the molecular basis by which these
distinct cis-acting structural elements mediate 43S
pre-initiation ribosome complex recruitment and
scanning. Similar to eIF4A, the selectivity by which
eIF4AIII, DDX3, DHX29 or RHA exert translational
control is governed by distinct interactions between
a defining cis-acting RNA element within the 5'
termini of an mRNA and its cognate DExH/D-box
RNA helicase. Together these select RNA-protein
interactions generate distinct trRNPs important for
targeted protein synthesis (Table 1). As discussed
below, the discrete composition and architecture
of each DExH/D-box RNA helicase trRNP confers
targeted roles for eIF4AIII, DDX3, DHX29 and
RHA in ribosome recruitment and scanning that
extends beyond basic mRNA unwinding to
encompass dynamic trRNP remodeling as well.
Its associations with eIF4G and the initiation
factors eIF4B and eIF4H coordinate this activity
near the 5' cap of an mRNA . eIF4G, by virtue
of its interaction with the cap-binding protein
eIF4E and a target transcript, recruits eIF4A to the
5' terminus of an activated mRNA [1, 2]. The
interaction of eIF4G with the helicase domain of
eIF4A stabilizes eIF4A in a closed, RNA-bound
conformation that stimulates its helicase activity
. This effect is also facilitated by eIF4B and
eIF4H, which function to regulate the affinity of
eIF4A for ATP and ADP during its ATPase-
driven RNA remodeling as well as its association
with a target mRNA . eIF4B and eIF4H also
have critical roles in preventing mRNA refolding
and for directing the movement of the 43S pre-
initiation ribosome complex in a 5' to 3' direction .
The necessity of eIF4A for translation initiation of
cellular mRNAs harboring 5' secondary structure,
which characterizes many eukaryotic transcripts,
together with the impact of its mutation on global
translation indicated eIF4A as a universal effector
of protein synthesis . This established eIF4A,
together with eIF4G, as an integral member of the
eIF4E trRNP complex regulating steady-state
expression of the majority of cellular transcripts
(discussed further in the section ‘The CBC and
eIF4E trRNPs’). However, a recent application of
ribosome profiling to the study of eIF4A-dependent
translation control revealed paradigm-shifting
specificity to eIF4A-mediated protein synthesis .
Ribosome profiling is a recently introduced technique
that has already revolutionized the study and
understanding of eukaryotic translation control by
allowing for an in vivo global characterization of
translational landscapes, such as that of eIF4A-
dependent translation control [15, 16]. By combining
polysome profiling with nuclease footprinting and
deep sequencing, this experimental approach
provides high-resolution data on both ribosome
abundance and positional occupancy along mRNAs
in live cells [15, 16]. The outcome is two-fold: (1)
the identification of mRNAs subjected to regulation
within a translational landscape based upon a change
in their number of ribosome footprints between
control and experimental conditions and (2) a
molecular basis for this effect based upon the
accompanied accumulation in ribosome footprint
RNA binding proteins target select mRNA templates for translation 111
DDX3 also functions in late-stage initiation with
targeted 80S ribosome formation . Here DDX3
coordinates a protein bridge or conformational
rearrangement within the 60S ribosomal subunit
that facilitates its recruitment to and association
with the 40S ribosomal subunit at the start AUG
to generate an elongation-competent 80S ribosome
. This effect is critical for expression of
hepatitis C virus in a manner that is independent
of cap-dependent scanning activity .
Experimental data also indicate a role for select
DDX3 trRNPs in inhibiting cellular cap-dependent
initiation by functioning as a competitive eIF4E-
binding protein and translational inhibitor .
DDX3 demonstrates a strong cellular affinity for
eIF4E, which impedes its association with eIF4G
. The outcome is impaired eIF4E trRNP
formation and repressed translation activity .
This effect is independent of DDX3’s ATPase
or helicase activity . However, the cellular
association of DDX3 with eIF4E and its significance
for regulating protein synthesis remains controversial
[21-23]. Likewise, the mRNA signatures and/or
protein factors that dictate the specificity, both in
composition and function, of the DDX3 trRNP in
targeted translation control remain ambiguous.
The DExH-box RNA helicase DHX29 is critical
for generating a trRNP that facilitates expression
of cellular mRNAs harboring stable stem-loop
structures within their 5' termini . DHX29
associates with the 40S ribosomal subunit within a
43S pre-initiation complex . It is positioned
near the mRNA entry channel latch where it
stimulates its opening to capture single-stranded
bases that have been released from resolution of
nearby stem-loop structure [27-29]. This activity
of DHX29 in conjunction with the known roles of
eIF4A in mRNA unwinding collaboratively facilitate
association and scanning of the 43S pre-initiation
ribosome complex along a target mRNA . The
outcome is ensured fidelity of base inspection and
start codon recognition [27, 30]. Thus, DHX29
drives critical trRNP remodeling rather than traditional
mRNA unwinding to facilitate efficient and
appropriate translation initiation [28, 30]. The
significance of this translation activity is seen in
the silencing of DHX29 expression, which results
in suppression of cellular translation by approximately
fifty percent . However, identification of the
eIF4AIII is a known component of exon junction
complexes, which are dynamic protein assemblies
that organize upon the coding sequence of a
mRNA with the conclusion of a splicing event to
facilitate critical post-transcriptional activities. A
recent study, however revealed the significance of
eIF4AIII as a 5' interacting factor necessary for
translation initiation of mRNAs harboring stable
stem loop structures within their 5' UTRs .
The recruitment of eIF4AIII to the 5' terminus of
a target transcript is facilitated by its direct
interaction with the 5' scaffolding factor CTIF and
is independent from its structural and functional
integration within exon junction complexes .
The ATPase/helicase function of eIF4AIII is
required for its stimulatory effect on translation
initiation, indicating a role for this DEAD-box
RNA helicase in resolving the stem-loop secondary
structures to facilitate 43S pre-initiation ribosome
complex association and scanning on target transcripts
. However, the mRNA features and/or protein
cofactors that contribute to the specificity of
the eIF4AIII trRNP in targeted 5'-cap-dependent
translation control remain to be elucidated.
For DDX3, its functions in translation initiation
are diverse and transcript-specific. In the case of
cellular mRNAs with long and highly structured
5' UTRs, DDX3 is critical for translation activity via
its helicase function and interaction with eIF4A
and PABP of an activated mRNA as well as eIF2
and eIF3 of the 43S pre-initiation ribosome complex
[19-21]. These identified cellular interactions indicate
significance for DDX3 in functioning synergistically
with eIF4A to resolve extensive secondary structure
in the 5' terminus, allowing for ribosome association
Similarly, DDX3 is necessary for resolving the
complex secondary structure directly near the
5' cap of the human immunodeficiency virus type
1 (HIV-1) genomic RNA . The outcome is
productive translation and generation of the major
viral structural protein Gag . Distinctly, however,
this requirement of DDX3 for targeted HIV-1
translation control is independent of its helicase
activity . Instead, it involves the association of
DDX3 with eIF4G, PABP, and the viral protein
Tat to generate a DDX3 trRNP that activates the
target HIV-1 genomic RNA for 43S pre-initiation
ribosome complex attachment and subsequent protein
112 Sarah Fritz & Kathleen Boris-Lawrie
RHA to a target transcript, whether it be through
direct PCE RNA binding or a protein bridge,
facilitates trRNP remodeling in a manner that
allows for 43S pre-initiation ribosome complex
recruitment, scanning, and ultimately target protein
production [32, 39, 40]. An unresolved issue,
however, is a complete characterization of the RHA
trRNP and how this informs its role in targeted
Collectively, the DExH/D-box RNA helicase family
harbors several members with critical roles in 43S
pre-initiation ribosome complex recruitment. Yet,
as discussed, the functional significances for each
DExH/D-box RNA helicase in this process are
distinct (mRNA unwinding versus trRNP remodeling)
to result in select trRNPs, like the RHA trRNP,
that afford the cell novel and diverse mechanisms
for controlling the initiation of protein synthesis.
Irrespective, an outstanding question across all
mechanisms of 43S pre-initiation ribosome complex
recruitment is the manner by which the mRNA
becomes positioned within the binding channel of
the 40S ribosomal subunit and the location at
which scanning-dependent base inspection begins
. Is the mRNA threaded through its binding
channel in the 40S ribosomal subunit or does the
43S pre-initiation ribosome complex undergo
direct positioning near the 5' terminus ? How
close to the 5' cap does the 43S pre-initiation
ribosome complex need to bind in order to engage
appropriate scanning in the correct reading frame
? It is likely that the molecular bases for these
effects are distinct for each class of transcripts and
are mediated by the particular mode of action of
the associated DExH/D-box RNA helicase.
The cap-dependent scanning mechanism of translation
initiation was identified as the defining feature for
eukaryotic protein synthesis . The study of
RNA viruses, however, soon challenged this central
canon with the observation of several alternative
mechanisms for translation initiation. These include,
but are not limited to, internal ribosome entry-site
(IRES)-mediated translation initiation, ribosome
shunt, ribosomal frameshifting, leaky scanning,
non-AUG initiation and reinitiation . Variations
to the central theme of 5'-end-dependent translation
were also soon realized amongst classes of cellular
molecular signatures, both mRNA sequence motifs
and protein cofactors, which characterize the specific
DHX29 trRNP and its translational landscape,
have yet to be defined.
DHX9/RNA helicase A (RHA) is critical for
facilitating the expression of retroviral transcripts,
including the human pathogenic retroviruses HIV-1
and HTLV-1, as well as the cellular proto-oncogene
junD [32-35]. Here, this DEIH-box helicase
selectively associates with the 5' cis-acting post-
transcriptional control element (PCE) that defines
these target mRNAs [32-35]. The PCE is a G-C-
rich, dual stem-loop structure that specifically
regulates cap-dependent translation of harboring
transcripts [33, 36-38]. Notably, the PCE functions
as a positive cis-acting translational regulator .
This effect is mediated through its association
with the trans-acting host factor RHA .
Suppression of cellular RHA expression reduces
PCE translation activity, the association of target
mRNAs with ribosomes, and subsequent protein
production [32-34]. The outcome, as studied thus
far, is impaired infectivity of HIV-1 progeny
The role of RHA in cap-dependent translation
control requires its association with the 5' terminal
PCE as well as its ATPase activity, indicating
canonical helicase function in its molecular basis
of protein synthesis [34, 35]. However, RHA can
also function in translation control independent of
PCE binding. Here RHA is recruited to a target
mRNA through association with a cognate RNA
binding protein. Such is the case for regulated
expression of the pluripotency factor Oct4 .
The RNA binding protein Lin28 recruits RHA to
the Oct4 mRNA, which in turn facilitates ribosome
association of the trRNP and subsequent protein
expression . A similar molecular basis for
RHA-mediated translation control is critical for
regulated expression of the type I collagen mRNAs
. The RNA binding protein La ribonucleoprotein
domain family member 6 specifically recognizes
and binds the defining 5' stem-loop structure of
type I collagen mRNAs to bridge an association
with RHA, stimulating translation activity .
Likewise, nuclear factor 90 bound to the 5' terminus
of p53 recruits RHA to this target mRNA and
facilitates its expression in response to DNA
damage . It is proposed that the recruitment of
RNA binding proteins target select mRNA templates for translation 113
translational regulation of HIV-1, IRES activity
Yet, the concept of end-independent translation
initiation is still well founded with unifying themes
observed across instances of accepted IRES activity.
This is particularly evident for IRES-mediated
translation control of RNA viruses. Type I and II
IRESes are identified by their maintained use of
eIF4G and eIF4A to bridge contacts with eIFs 2
and 3 of the 43S pre-initiation ribosome complex,
which facilitates its recruitment to a target mRNA
[1, 43]. Instead of associating with eIF4E at the 5'
cap, as seen in canonical cap-dependent initiation,
eIF4G and eIF4A in these instances associate with
RNA elements within the IRES itself . A type I
IRES, as exemplified in poliovirus, is mechanistically
distinguished from a type II, such as the encephalo-
myocarditis virus IRES, by the fact that decent
scanning of the 43S pre-initiation ribosome complex
to the start codon is still involved upon mRNA
association [1, 43]. On the contrary, type II IRESes
exhibit recruitment of the 43S pre-initiation complex
in close proximity to the start codon with minimal
scanning observed [1, 43]. A third class of IRES,
type III, is identified by the requirement of only
eIFs 2, 3 and 5 to facilitate direct positioning of
the 43S pre-initiation ribosome complex at the
start codon [1, 43]. This is observed in the case
of hepatitis C virus-mediated translation control
[1, 43]. Type IV IRESes, like the cricket paralysis
virus, interact with the ribosome independently of
any eIF and initiate translation via a structural
mimic of the initiator methionyl-tRNA conferred
by the IRES itself [1, 43].
RNA binding proteins are also critical to the
mechanisms by which IRESes initiate translation
by binding directly to or nearby an IRES to regulate
its efficiency [50, 51]. Known as IRES trans-acting
factors (ITAFs), these RNA binding proteins are
critical to the diversification observed in IRES
activity, both in mechanism and occurrence [50, 51].
They can function in RNA conformation control
or in RNP remodeling by affecting the stability of
RNA secondary structure or serving as bridging
cofactors with the ribosome, respectively [50, 51].
Of the ITAFs identified thus far, many are members
of the heterogeneous nuclear ribonucleoprotein
(hnRNP) family and include PTB (hnRNP1),
hnRNPA1, hnRNPE2, hnRNPE1, hnRNPC and
transcripts. This was particularly observed during
times of cell stress and division, resulting in non-
canonical translation initiation becoming a hallmark
feature of the cellular stress response and mitosis
[44-47]. Recent application of ribosome profiling
to the study of eukaryotic translation control has
extended our realization of alternative initiation
mechanisms in cells . The data from these studies
support pervasive engagement of non-AUG codons
and upstream open reading frames in the steady-
state regulation of cellular protein synthesis .
Thus, eukaryotic translation initiation encompasses
a diverse array of mechanisms to regulate the start
of protein synthesis and intricately control gene
Internal ribosome entry-site (IRES)-mediated
translation initiation is by far the most studied
mechanism of alternative engagement into protein
synthesis. An IRES is an RNA element capable of
directly recruiting the small ribosomal subunit to
a start codon without the need for interactions
with the 5' cap or cap-associated factors [1, 43,
48]. Although pervasively observed to control the
expression of many RNA viruses and stress-response
transcripts, there is no unifying mechanism to
describe IRES-mediated translation control [1, 43,
48]. This is because an IRES can only be identified
experimentally due to the lack of sequence and
structure conservation . The gold-standard
approach is a bicistronic reporter assay whereby a
putative IRES element is cloned to regulate the
expression of a second, internal cistron within a
plasmid harboring two adjacent open reading
frames (typically Renilla and firefly luciferase)
. Expression of the first cistron is cap-dependent
whereas protein production from the second
cistron is regulated by the putative IRES element
. A relative increase in second cistron protein
production, when compared to a construct cloned
with a random sequence in its place, indicates
IRES activity . A major caveat of this approach,
however, is the challenge of discriminating true
IRES activity from cryptic promoter functions
. Additionally, the manner by which a putative
IRES sequence is cloned out from its original
context and into the bicistronic reporter can have
significant consequences on the classification of a
particular RNA element as IRES or not .
Thus, in many instances, such as that for the
114 Sarah Fritz & Kathleen Boris-Lawrie
The peptidyl transferase center is a region of
highly conserved ribosomal RNA that functions in
orienting the acceptor stems of the A- and P-site
tRNAs, which are the regions bound to the
cognate amino acids, to drive the two-step
reaction that results in peptide bond formation
. This amino acid linkage between the A- and
P-site tRNAs triggers the ribosomal subunits to
move in a ratchet-like motion that results in a
hybrid tRNA state prior to complete translocation
. Here the acceptor stems of the A- and P-site
tRNAs are positioned within their adjacent E- and
P-sites, respectively, while their anticodon stem
loops remain in their corresponding A- and P-sites
. Complete translocation of the tRNA-ribosome
complex to the subsequent codon is driven by
eEF2 association, GTP-hydrolysis and phosphate
release  (Figure 2). This results in a deacylated
tRNA (a tRNA without its charged amino acid or
associated polypeptide chain) within the 5' E-site,
a P-site tRNA with a dipeptide bound to its acceptor
stem, and an unoccupied A-site that is primed for
acceptance of the next aminoacyl-tRNA based
upon anticodon-codon base pairing. Repetition of
these three steps along an open reading frame
grows the encoded polypeptide and occurs until a
stop codon is reached, which triggers termination
and release of the protein product.
Intricate to the control of translation elongation
are select RNA binding proteins, which associate
with target transcripts and form distinct trRNPs
that regulate the dynamics of polypeptide synthesis.
Four major elongation effectors have been identified
and mechanistically studied. They are, the fragile
X mental retardation protein (FMRP), heat shock
protein 70 (Hsp70), pumilio and argonaute (PUF-
Ago), and heterogenous ribonucleoprotein E1
(hnRNP E1) [54-58]. Although their targets and
mechanisms for regulation are distinct, the unifying
principle among all four effectors is that their
formation of distinct trRNPs is intricate to the
molecular basis by which they regulate the dynamics
of polypeptide synthesis.
FMRP binds the mRNA coding sequence of pre-
and postsynaptic transcripts and induces elongation
pausing by competing with P-site tRNA binding
[54, 55]. The result is impacted ribosome-tRNA
dynamics that compromise the elongation process
on target transcripts [54, 55]. This directed translation
hnRNPL [50, 51]. This significance is linked with
the nuclear-cytoplasmic shuttling activity and
additional post-transcriptional functions of hnRNPs,
and appears distinct for each IRES target .
Thus, the formation of select trRNPs to regulate
eukaryotic translation is fundamental to IRES-
The importance of trRNPs in translation control
is also seen for the other mechanisms of end-
independent initiation. Ribosome shunt, ribosomal
frameshifting, leaky scanning, non-AUG initiation
and reinitiation are observed due to trRNP elements
directing the ribosome to move in an alternative
manner as their names imply . Although
mechanistically characterized by variations in
RNA sequence and/or structure impacting ribosomal
movement, it can be envisioned that distinct RNA
binding proteins have critical roles in these
processes as well.
Extension of a polypeptide chain to produce a
full-length protein product occurs by three steps:
amino acid incorporation, peptide bond formation
and ribosome translocation. Two elongation factors
(eEFs) coordinate these activities, the dynamics of
which are influenced by associated RNA binding
proteins. Thus, trRNP biology is fundamental to
targeted translation control during the elongation
stage of protein synthesis.
Polypeptide extension begins with the incorporation
of a charged transfer RNA (tRNA) into the
unoccupied 3' aminoacyl-site (A site) of an 80S
ribosome poised to elongate from the start codon
 (Figure 2). This activity is facilitated by the
multi-subunit complex eEF1 in its GTP-bound form
. Anticodon-codon base pairing drives the
identification of appropriate aminoacylated tRNA
incorporation . Only correct matches allow for
conformational arrangements of the tRNA that are
tolerated by the ribosome . This recognition
triggers GTP hydrolysis, eEF1 release, and full
accommodation of the accepted aminoacyl-tRNA
in the A site .
Next, the A-site aminoacyl-tRNA is linked to the
P-site aminoacyl-tRNA, which harbors the initiator
methionine, via peptide bond formation 
(Figure 2). This reaction is catalyzed by the peptidyl
transferase center of the large ribosomal subunit .
emersion from the ribosomal exit tunnel . The
factors directing this specificity and its significance
for mRNA-mediated translation control remain to
be elucidated. hnRNP E1, on the other hand, regulates
eEF1A function by binding to and inhibiting
its dissociation from ribosomes . The outcome
is impaired ribosome translocation and protein
production . This effect is critical for regulated
expression of epithelial-mesenchymal transition
transcripts that are significant in development and
The translation process concludes with termination
of polypeptide elongation, nascent chain release,
and ribosome recycling. Translation termination is
triggered by the recognition of a stop codon
(UAA, UAG or UGA) within the A-site of an
elongating ribosome  (Figure 3). This recognition
occurs in eukaryotes by the eukaryotic termination
factor 1 (eRF1), which structurally exists as a tRNA
mimic and associates with the stop codon-containing
A site . eRF1 harbors a conserved GGQ motif
that positions within the peptidyl transferase center
and induces conformational changes that allow
access of a water molecule to this active site 
(Figure 3). The result is breakage of the ester
bond that holds the polypeptide chain to the P site
tRNA, releasing the protein product . This
function of eRF1 is stimulated by its association
with the second termination factor, eRF3 . eRF3
is a GTPase whose affinity for GTP is increased
by association with eRF1 . eRF1 and eRF3 are
found within stable complexes and their ribosome
association triggers GTP hydrolysis, movement of
the GGQ motif into the peptidyl transferase center,
and eRF1-induced polypeptide release .
The ATP-binding cassette protein ABCE1 is
responsible for ribosome recycling upon nascent
chain release  (Figure 3). The retention of
eRF1 on post-termination complexes recruits ABCE1
and stimulates its ATPase activity . Hydrolysis
of ATP induces a conformational switch in ABCE1
from a closed ATP-bound state to an open ADP-
bound state . This movement of ABCE1
causes the 60S ribosomal subunit to split away
from its 40S counterpart . Initiation factors
eIF1, 1A and 3 facilitate subsequent deacylated
tRNA and mRNA release from the 40S ribosomal
control is critical for the spatiotemporal regulation
of neuronal protein expression, which is essential
for proper nervous system function [54, 55]. The
specificity for FMRP-mediated control of translation
elongation is driven by its affinity for particular RNA
sequence elements, GAC, ACUG/U and A/UGGA,
which have predicted G-quadruplex secondary
structure . Mutational studies indicate that the
ability of FMRP to bind RNA is sufficient for its
induced elongation pausing effect [54, 55].
Hsp70 is a critical molecular chaperone that
facilitates protein production by associating with
nascent polypeptide chains during the elongation
process and coordinating their correct folding into
a functional protein product . This activity of
Hsp70 involves its association with ribosomal
proteins of the peptide exit tunnel, particularly the
large ribosomal subunits RPL4 and RPL22, and
elongation factor eEF1A . These interactions
function to facilitate efficient movement of the
nascent polypeptide chain through the peptide exit
tunnel and proper elongation kinetics . Heat
shock, however, alters the interaction of Hsp70
with the ribosome and eEF1A such that the peptide
exit tunnel becomes constricted and the nascent
polypeptide chain exposed in a manner that
compromises elongation kinetics to cause global
ribosome pausing at codon 65 of most mRNAs
. Codon 65 correlates with a sixty-five amino
acid polypeptide, which is long enough to traverse
the peptide exit tunnel and become exposed so
that it is affected by altered interactions of Hsp70
during heat shock. The outcome is regulated
translation that is fundamental to the heat shock
PUF-Ago and hnRNP E1 control translation
elongation by regulated interactions with elongation
factor eEF1A. eEF1A is the subunit of eEF1 that
binds GTP and is responsible for delivering
aminoacyl-tRNAs to the A site of the ribosome
. In the case of PUF-Ago, these RNA binding
proteins associate with eEF1A in a manner that
inhibits its GTPase activity . The effect is
stalled elongating ribosome complexes within the
open reading frames of transcripts, hindering protein
production . The influence of PUF-Ago on
eEF1A GTPase activity appears specific for
elongating complexes that have traversed the
mRNA to the point of nascent polypeptide chain
RNA binding proteins target select mRNA templates for translation 115
Two main exceptions to this mechanistic paradigm
of eukaryotic translation termination are known.
They occur during the phenomena of reinitiation
and nonsense-mediated mRNA decay. Reinitiation
is when the ribosome fails to dissociate from a
transcript upon termination of polypeptide synthesis
. Instead it resumes scanning to a downstream
subunit via competitive binding interactions .
The outcome is recycling of all factors required
for subsequent rounds of protein synthesis.
Interactions between the 3' poly-A binding protein
(PABP) and the 5' scaffolding factor eIF4G,
which as previously discussed function to create a
circularized mRNA, allow for efficient reinitiation
and subsequent rounds of protein synthesis upon
116 Sarah Fritz & Kathleen Boris-Lawrie
Figure 2. Schematic of canonical eukaryotic translation
elongation. Canonical eukaryotic translation elongation
proceeds in three steps: amino acid incorporation, peptide
bond formation, and ribosome translocation. Upon formation
of an elongation-competent 80S ribosome at the start
codon (AUG), the elongation factor eEF1 facilitates
association of a charged tRNA with an available codon
in the adjacent, 3' A-site. Recognition of appropriate
anticodon-codon base pairing stimulates GTP hydrolysis
and release of eEF1. Peptide bond formation is subsequently
catalyzed by the peptidyl transferase center of the large
ribosomal subunit. This results in a polypeptide chain
of n+1 positioned with the A site of the ribosome.
Ribosomal translocation to the next 3' codon is stimulated
by peptide bond formation and completed with the
association of elongation factor eEF2 and GTP hydrolysis.
Repetition of these three steps along an open reading
frame grows the encoded polypeptide.
Figure 3. Schematic of canonical eukaryotic translation
termination. Canonical eukaryotic translation termination
is triggered by the recognition of a stop codon (e.g.
UAA) within the A-site of an elongating ribosome. This
recognition occurs by the eukaryotic translation termination
factor eRF1 in complex with eukaryotic translation
termination factor eRF3 and GTP. eRF1 harbors a
conserved GGQ motif that positions within the peptidyl
transferase center upon stop codon recognition and GTP
hydrolysis. This movement of the GGQ motif into the
peptidyl transferase center induces a conformational
rearrangement in the large ribosomal subunit that causes
hydrolysis of the ester bond linking the polypeptide chain
to the P site tRNA, releasing the protein product. The
ATP-binding cassette protein ABCE1 resolves the post-
termination complex by splitting away the large (60S)
ribosomal subunit from the now vacant 80S complex.
Initiation factors eIF1, 1A and 3 facilitate subsequent
separation and recycling of the deacylated tRNA,
mRNA, and small (40S) ribosomal subunit.
AUG where a second initiation event is engaged .
This is observed among cellular transcripts that
harbor short upstream open reading frames and is
critical for their targeted translation control .
A principle of the eukaryotic scanning model of
protein synthesis is that a 43S pre-initiation ribosome
complex scans an mRNA until the first AUG is
detected . It will then engage in 80S formation
and polypeptide production irrespective of the
length of the open reading frame . Consequently,
for mRNAs that harbor several open reading frames,
a competition arises between upstream initiation
events and downstream polypeptide production
. Often it is only when translation activity
is impaired, such as during cell stress, does
compromised initiation at the upstream open reading
frame allow for effective expression of the
downstream protein product (see section below,
‘Regulation of eukaryotic translation’). This results
in a means for effective translation control, as the
downstream open reading frame often encodes the
functional cellular protein.
Reinitiation is also intricate to the molecular basis
by which many infectious viral proteins are expressed
[43, 61]. Although the mechanisms regulating
reinitiation are diverse, a common theme is the
role of distinct trRNPs in mediating this effect.
Such is the case, for example, in the expression of
critical structural proteins for mammalian caliciviruses,
which are responsible for several life-threatening
diseases in animals. An RNA element termed
‘termination codon upstream ribosome-binding
site’ (TURBS) is critical to reinitiation and expression
of infectious downstream viral protein products on
caliciviral transcripts . The TURBS consists of
two motifs that harbor sequences of the 3' terminus
of the upstream open reading frame and the region
between the first stop and second start site .
These motifs are necessary for reinitiation events
by functioning to capture 40S ribosomal subunits
upon termination of upstream translation, effects
that occur by the TURBS element forming a
critical trRNP that associates with eIF3 and
mimics 18S rRNA . The outcome is effective
expression of downstream viral protein products
in measured amounts that are critical to caliciviral
Nonsense-mediated mRNA decay is the essential
cellular process of mRNA surveillance and quality
control. It is responsible for the resolution of
premature termination events and subsequent
degradation of the effected mRNA so as to prevent
production and accumulation of rogue protein
products. Premature termination results from the
recognition of premature termination codons by
eRF1 and eRF3 . Premature termination codons
are stop codons that are positioned upstream of
the normal stop codon within an open reading
frame . Although eRF1 and eRF3 are recruited
to premature termination codons, the subsequent
termination events of ABCE1 recruitment and
ribosome recycling do not occur. Instead, an
alternative trRNP is formed that halts the translation
process, inhibits subsequent initiation events, and
induces mRNA decay . This alternative trRNP
consists of a complex series of interactions between
the cap-binding protein CBP80/20, a nearby exon
junction complex, and recruited factors from the
up-frameshift (UPF) and suppressor of morphogenetic
effect on genitalia (SMG) protein families .
Collectively, these interactions and the progression
in their association regulate trRNP dynamics that
result in altered termination fates to control protein
trRNPs in the regulation of eukaryotic
Global and targeted regulation of eukaryotic
translation is observed at all three stages of protein
synthesis. These mechanisms can be constitutive
or induced upon changes in the cellular environment.
In either case, distinct trRNP formation and activity
is fundamental to each effect.
43S pre-initiation ribosome complex formation,
its recruitment and scanning, and 60S ribosomal
subunit joining are all examples of targetable
steps for regulated gene expression at the stage
of translation initiation. As previously discussed,
DExH/D-box RNA helicases and the select trRNPs
they create, are instrumental for coordinating 43S
pre-initiation ribosome complex recruitment and
scanning on target transcripts. Other trRNPs, formed
by alternative RNA binding proteins, contribute a
similar effect. Two classic examples are the regulated
expression of ferritin mRNA in response to iron
homeostasis and male-specific lethal-2 (msl-2)
translation in Drosophila X-chromosome dosage
compensation. During iron deprivation, the RNA
RNA binding proteins target select mRNA templates for translation 117
binding protein iron regulatory protein (IRP)
binds with high affinity to its cognate cis-acting
RNA element, the iron-responsive element (IRE),
which defines the 5' terminus of ferritin mRNA
. The IRE is a stem-loop structure that when
bound by IRP effectively impedes 43S pre-initiation
ribosome complex association and expression of
the ferritin mRNA [63, 64]. Elevated iron levels
reduce the affinity of IRP for IRE, effectively
lifting its block on translation initiation and allowing
for expression of ferritin .
Inhibition of msl-2 expression in female flies is
fundamental to dosage compensation and survival.
This effect is mediated by the female-specific
RNA binding protein Sex-lethal (SXL), which
binds to distinct poly-uridine tracts in the 5' and 3'
termini of msl-2 and inhibits translation . The
molecular basis for this effect is two-pronged. At
the 3' terminus, SXL impairs 43S pre-initiation
ribosome complex recruitment by associating with
the 3'-bound poly-A binding protein (PABP) to
interfere with dynamics at the 5' cap that allow for
appropriate initiation [65, 66]. This coordination
between 3' effectors and a 5' outcome is mediated
by PABP-induced mRNA looping . At the
5' terminus, SXL stalls scanning 43S pre-initiation
ribosome complexes that were able to circumvent
the 3'-mediated block, effectively reducing their
affinity for msl-2 and causing their dissociation
. The outcome is repressed expression of msl-2
protein and effective dosage compensation.
Blockade of 60S ribosomal subunit joining is
another effective mechanism employed by distinct
trRNPs to coordinate targeted translation regulation.
Such is the case for controlled expression of erythroid
15 lipoxygenase (LOX) mRNA during erythroid
differentiation. LOX mRNA encodes a critical
enzyme important for internal membrane
reorganization during late stages of red blood cell
maturation [67, 68]. Temporal restriction of its
expression is mediated by the association of the
RNA binding proteins hnRNP K and hnRNP E1
with the cis-acting differentiation control element
(DICE) in the 3' terminus of the LOX mRNA [67,
68]. DICE is a repeated CU-rich motif that when
bound by hnRNP K and hnRNP E1 inhibits 60S
ribosomal subunit joining by interfering with
initiation factor activity that mediates this effect
[67, 68]. A similar molecular basis is seen in the
spatiotemporal regulation of β-actin expression
whereby the RNA binding protein Zipcode
binding protein 1 (ZBP1) binds the 3' cis-acting
zipcode RNA element in the β-actin mRNA and
impedes 60S ribosomal subunit joining to
effectively suppress translation activity . This
blockade is uplifted by phosphorylation of ZBP1,
which reduces its affinity for the β-actin mRNA
and allows translation to proceed .
Regulated elongation is the third effective means
by which protein synthesis can be controlled. As
previously discussed, there are known instances
for targeted elongation regulation in which
specific RNA binding proteins influence trRNP
dynamics to control elongating ribosome activity.
Global regulation of elongation is also observed
and it occurs with the phosphorylation of the
elongation factor eEF2. This post-translational
modification interferes with eEF2-GTP complex
formation, which inhibits the association of eEF2
with the ribosome and effectively impedes
Phosphorylation of eEF2 occurs by the eEF2
kinase . Mammalian eEF2 kinase activity is
controlled by the cell’s central commander of
translation: the mammalian target of rapamycin
(mTOR) (Figure 4). mTOR is a serine/threonine
kinase that directs protein synthesis in reflection
of the cellular environment by acting upon signals
received from almost all major cell-receptor
signaling pathways. These include, but are not
limited to the PI3K/AKT and Ras/ERK signaling
cascades . One downstream effector of mTOR
is the ribosomal protein S6 kinase (S6K) (Figure 4).
During normal growth conditions or in response
to mitogenic stimuli, mTOR is activated and
phosphorylates S6K . S6K, in turn,
phosphorylates the eEF2 kinase, which results in
eEF2 kinase inactivation, eEF2-GTP association,
and the promotion of translation elongation [70,
72]. However, when mTOR is inactivated by cellular
stress the inhibitory block of S6K on eEF2 kinase
activity is relieved, resulting in phosphorylation of
eEF2 and impaired translation elongation activity
[70, 72]. The outcome is suppression of global
Regulated protein synthesis in response to the
environment is also observed at the stage of
translation initiation. Here mTOR and its downstream
118 Sarah Fritz & Kathleen Boris-Lawrie
activity results in hypophosphorylation of 4E-BP1,
a strong 4E-BP1:eIF4E association, and consequent
suppression of eIF4E-dependent translation initiation
Besides directing mTOR-mediated translational
control, the cellular environment influences protein
synthesis by regulating the function of eIF2 in the
earliest stage of initiation, 43S pre-initiation ribosome
complex formation. The very initial association of
an initiator methionyl-tRNA with a 40S ribosomal
subunit is facilitated by eIF2 in its GTP-bound
form [2, 3]. eIF2 consists of 3 subunits: α, β and γ
[2, 3] (Figure 6). The α subunit together with β
serves as a critical allosteric effector of direct
GTP and initiator methionyl-tRNA binding to the
γ subunit of eIF2 [2, 3]. GTP association occurs
first and is rate-limiting for initiator methionyl-
tRNA binding [2, 3]. Phosphorylation of the α
subunit at serine residue 51 inhibits the exchange
of GDP for GTP on eIF2, an effect mediated by
the guanine nucleotide exchange factor eIF2B
[2, 3] (Figure 6). The outcome is impaired efficiency
of ternary complex formation (initiator methionyl-
tRNA and eIF2 bound to the 40S ribosomal
subunit), which results in reduced 43S pre-initiation
ribosome complex formation, its recruitment to
mRNAs, and subsequent translation [2, 3].
Four distinct protein kinases have been identified
that phosphorylate eIF2α: haem-regulated inhibitor
kinase (HRI), protein kinase R (PKR), PKR-like
endoplasmic reticulum kinase (PERK), and general
control non-derepressible-2 (GCN2) (Figure 6).
Notably, their activation occurs in response to a
variety of environmental stressors as the cell attempts
to rapidly adjust to a change in its homeostasis:
Iron deficiency and osmotic or heat shock activate
HRI; double-stranded RNA triggers PKR activity;
ER-stress and hypoxia activate PERK; amino-acid
deprivation and UV irradiation stimulate GCN2
. The outcome in all instances is phosphorylation
of eIF2α and suppression of general translation, as
the 43S pre-initiation complex is fundamental to
protein synthesis. However, distinct transcripts
like mammalian activating transcription-factor-4
(ATF4) and the yeast transcriptional activator
GCN4 circumvent this block and exhibit efficient
expression even in the advent of eIF2α
phosphorylation. This targeted translation activity
is specific for critical stress response proteins and
effector S6K once again serve as a central line of
command to influence trRNP dynamics and protein
production (Figure 4). In this instance, mTOR-
directed S6K activation targets the translation
initiation factor eIF4B and the regulatory protein
programmed cell death 4 (PDCD4) [71, 72].
Phosphorylation of eIF4B by S6K enhances eIF4B’s
stimulation of eIF4A helicase activity and protein
production [71, 72]. Likewise, phosphorylation of
PDCD4 results in eIF4A-mediated translational
activation by causing its subsequent ubiquitylation
and degradation [71, 72]. PDCD4 is an inhibitor
of eIF4A helicase activity; thus, its removal relieves
an inhibitory block on eIF4A-dependent translation
initiation [71, 72]. The outcome of both effects is
activation of eIF4A and effective translation initiation
on target mRNAs.
Several additional downstream targets of S6K are
also known and their activity has significant
implications for regulated translation initiation
(Figure 4). These S6K targets are, the 40S ribosomal
subunit protein S6 (rpS6), the S6K1 Aly/REF-like
target (SKAR), the cAMP-responsive element
modulator τ (CREM τ), CBP80 of the CBP80/20
cap-binding protein complex, the proapoptotic
protein BAD, insulin receptor substrate IRS (IRS),
and mTOR itself . However, in many of
these studied instances, such as rpS6 and CBP80
phosphorylation, it remains controversial about the
exact effects of this post-translational modification
on their role in translation control and the influence
of S6K in these outcomes .
A second line of command used by mTOR to
control translation initiation activity is the eIF4E
inhibitory protein, 4E-binding protein 1 (4E-BP1)
[71, 72] (Figures 4 and 5). 4E-BP1 influences
cap-dependent translation initiation by competing
with eIF4G for eIF4E association [73-75]. A 4E-
BP1:eIF4E interaction effectively disrupts cap-
associated trRNP dynamics to suppress translation
initiation [73-75]. The association of 4E-BP1 with
eIF4E is regulated by its phosphorylation status.
In response to mitogenic stimuli, activation of mTOR
results in its hyperphosphorylation of 4E-BP1 [71,
76]. In this post-translational state, 4E-BP1 has
reduced affinity for eIF4E . This allows the
eIF4E cap-binding protein to interact with eIF4G
and promote cap-dependent translation . On the
contrary, stress-induced suppression of mTOR
RNA binding proteins target select mRNA templates for translation 119
The outcome is inhibited expression of ATF4
. However, in ER stress when misfolded proteins
accumulate and activate PERK, phosphorylation
of eIF2α causes the limiting pool of 43S pre-
initiation ribosome complexes to scan through
upstream open reading frame 2 and initiate translation
at the downstream ATF4 open reading frame .
The result is efficient synthesis of ATF4, which is
a major transcription factor that facilitates expression
of genes critical to resolving ER stress . A
similar molecular basis explains the translational
activation of yeast GCN4 in response to amino
acid starvation .
is fundamental to the cell’s ability to regain
homeostasis and avoid apoptosis and cell death.
The ability of specific mRNAs to engage in
translation in the advent of eIF2α phosphorylation
is dictated by their distinct trRNPs. In the case of
ATF4, its 5' terminus is characterized by two
upstream open reading frames . In cellular
states when eIF2α phosphorylation is low and
ternary complex is abundant, upstream open reading
frame 2 preferentially engages 43S pre-initiation
ribosome complexes in recruitment and scanning
over the downstream ATF4 open reading frame .
120 Sarah Fritz & Kathleen Boris-Lawrie
A hallmark feature of HIV-1 infection is global
suppression of host cell translation . This
effect is mediated by HIV-1-induced activation of
4E-BP1 . 4E-BP1 is the eIF4E inhibitory protein
that binds to the translation initiation factor eIF4E
and disrupts associated trRNP dynamics to impede
canonical cap-dependent protein synthesis. Since
the eIF4E trRNP is responsible for the bulk of
cellular steady-state protein synthesis (see below),
the outcome is observed global suppression of
host cell translation . Yet during this effect,
HIV-1 maintains expression of its critical structural
proteins . As an obligate parasite, HIV-1 requires
the host cell translation machinery for expression
of its encoded viral proteins. Furthermore, it does
Distinct mechanisms of translation regulation, both
global and specific, are also observed in virus-
infected cells. Here a complex interplay between
translation suppression and activation, and canonical
and non-canonical protein synthesis is essential
for viral replication and the host innate defense.
Although the specific mechanisms characterizing
this translational reprogramming event are diverse
among each virus infection, the central theme of
trRNP-mediated translation control is fundamental
to explaining each observed outcome. Human
immunodeficiency virus type 1 (HIV-1) infection
is a model example to demonstrate how trRNP
biology is at that core of translational reprogramming
that characterizes viral infections.
RNA binding proteins target select mRNA templates for translation 121
Legend to Figure 4. Overview of mTOR-directed eukaryotic translation control. Mammalian target of rapamycin
(mTOR) is a serine/threonine kinase that serves as the central commander of eukaryotic translation control. It directs
protein synthesis in reflection of the cellular environment by acting upon two downstream effectors: ribosomal
protein S6 kinase (S6K) and eIF4E binding protein 1 (4E-BP1). S6K targets several factors critical in the regulation
of translation. These include, the 40S ribosomal subunit protein S6 (rpS6), eEF2 kinase (eEF2K), CBP80 of the
CBP80/20 cap-binding complex, the S6K1 Aly/REF-like target (SKAR), regulatory protein programmed cell death 4
(PDCD4), and translation initiation factor eIF4B. The phosphorylation of these translation factors by mTOR-S6K
signaling alters their function in a manner that facilitates translation activity. For eEF2K, its phosphorylation by S6K
results in its inactivation. This allows elongation factor eEF2 to effectively associate with elongating ribosomes and
facilitate their translocation. Similarly, the phosphorylation of PDCD4 results in its inactivation to facilitate steady-
state protein synthesis. Here S6K-mediated phosphorylation of PDCD4 induces its ubiquitinylation and subsequent
degradation. This removes PDCD4 as an inhibitor of eIF4A helicase activity, allowing translation initiation to
proceed. Likewise, the phosphorylation of eIF4B by S6K also facilitates effective steady-state protein synthesis by
stimulating eIF4A helicase activity. In this instance, phosphorylated eIF4B exhibits enhanced association for eIF4A,
which effectively stimulates translation activity. The direct effects of mTOR/S6K-induced phosphorylation on rpS6
and CBP80 translation activity remain ambiguous; however, data support a stimulatory outcome on initiation. For
the second line of command, mTOR-directed 4E-BP1 signaling controls translation by regulating trRNP dynamics.
Here phosphorylation of 4E-BP1 by mTOR reduces its affinity for the eIF4E cap-binding protein. This allows eIF4E
to effectively associate with the translation initiation factor eIF4G and create a trRNP that is productive for steady-
state protein synthesis.
Legend to Figure 5. Regulation of 4E-BP1 phosphorylation events and its role in eukaryotic translation control.
The eIF4E binding protein 1 (4E-BP1) is a critical effector of translation activity. Its function is dependent on
upstream signaling events that converge on the regulation of its major commander, the mammalian target of
rapamycin (mTOR). In the presence of mitogenic stimuli (e.g. growth factors), mTOR is activated and results in the
hyperphosphorylation of 4E-BP1. The hyperphosphorylation of 4E-BP1 is a hierarchical event with phosphorylation
at threonine (Thr) residues 37 and 46 priming for phosphorylation at Thr70, which then allows for phosphorylation
at serine (Ser) residue 65. Phosphorylation at Ser65 is the critical effector post-translational modification that
regulates the functional effects of 4E-BP1 in translation control. In its presence, 4E-BP1 exhibits reduced affinity for
the eIF4E cap-binding protein. This allows eIF4E to associate with the translation initiation factor and scaffolding
protein eIF4G. An eIF4E-eIF4G association allows for effective trRNP formation that facilitates translation activity.
In the advent of stress, however, mTOR activity is reduced to result in hypophosphorylation at Ser65. This change in
the post-translational modification status of 4E-BP1 increases its affinity for eIF4E. A 4E-BP1-eIF4E association
effectively inhibits an interaction between eIF4E and eIF4G. This results in impaired trRNP formation and inhibited
translation activity. It has also been shown that phosphorylation at Ser101 of 4E-BP1 regulates phosphorylation at
Ser65 and that phosphorylation at Ser112 facilitates release of 4E-BP1 from eIF4E.
of regulation by 4E-BP1 and is known to be
functional during states of cell stress (see below).
Thus, the CBC trRNP provides an alternative
mechanism for HIV-1 viral protein expression
that is cap-dependent but independent of eIF4E
tRNP activity . Therefore, the translation
dynamics during HIV-1 infection demonstrate
how distinct trRNP activity choreographs targeted
so in an arguably cap-dependent scanning manner
[33, 34]. Yet, how is this possible with its suppression
of eIF4E trRNP activity? The answer is in the
ability of HIV-1 transcripts to engage within an
alternative trRNP that harbors the CBP80/20 cap-
binding protein complex (CBC) . CBC functions
in translation initiation like eIF4E, however, its
prominence is during the pioneer rounds of translation
(see below). Notably, CBC activity is independent
122 Sarah Fritz & Kathleen Boris-Lawrie
polypeptide synthesis. This effect is directed by
trRNP complexes, and in particular the defining
cap-binding proteins, CBP80/20 (CBC) or eIF4E.
Although CBC and eIF4E direct the interaction of
an mRNA with the ribosome in a similar manner,
the molecular basis by which each mediates this
effect is distinct . Consequently, the CBC trRNP
and the eIF4E trRNP have distinct functional
significances in the choreographing of eukaryotic
translation (Figure 7).
The CBC and EIF4E trRNPS
Two core trRNPs characterize general eukaryotic
cap-dependent translation. These are, the CBC trRNP
and the eIF4E trRNP. As previously discussed,
initiation of eukaryotic translation is defined by a
cap-dependent scanning mechanism whereby the
ribosome binds to the 5' terminus of an mRNA
and proceeds along the transcript, inspecting base-
by-base, for an appropriate start codon to initiate
RNA binding proteins target select mRNA templates for translation 123
Legend to Figure 6. Integration of stress response signals into the phosphorylation of eIF2α and its outcome
on regulated eukaryotic protein synthesis. Integral to the function of translation initiation factor eIF2 is its ability
to function as a GTPase. eIF2-GTP drives ternary complex formation and is necessary for 43S pre-initiation
ribosome complex assembly, recruitment and scanning. Start codon recognition triggers GTP hydrolysis, an outcome
that induces conformational rearrangements that halt the scanning process and engage 80S ribosome complex
formation. The exchange of GDP for GTP on eIF2 is necessary for subsequent re-engagement of eIF2-driven
translation initiation. This requires the guanine nucleotide exchange factor eIF2B, an effect that is highly regulated
by the cellular environment. eIF2 consists of 3 subunits: α, β and γ. The α subunit is the critical allosteric effector of
direct GTP binding. Phosphorylation of the α subunit at serine (Ser) residue 51 inhibits the exchange of GDP for
GTP on eIF2. This effectively impedes eIF2-mediated initiation events, resulting in compromised translation
activity. Phosphorylation of eIF2α occurs by four distinct kinases, each of which is stimulated by distinct cellular
stressors. Haem-regulated inhibitor kinase (HRI) is activated by iron deficiency and osmotic or heat shock, protein
kinase R (PKR) is stimulated by double-stranded RNA, PKR-like endoplasmic reticulum kinase (PERK) responds to
ER-stress and hypoxia, and general control non-derepressible-2 (GCN2) is activated by amino-acid deprivation and
UV irradiation. The outcome in all instances is phosphorylation of eIF2α, which impairs the exchange of GDP for
GTP to result in suppression of general translation.
Legend to Figure 7. Model of CBC and eIF4E trRNP dynamics in the control of eukaryotic translation.
Canonical eukaryotic cap-dependent translation is governed by a complex interplay between the CBC trRNP and the
eIF4E trRNP. Beginning at transcription, the CBP80/20 cap-binding protein complex (CBC) directly binds the 5' 7-
methyl-guanosine cap of a nascent transcript (1). This association is driven by the high affinity of CBC for 7-methyl-
guanosine and its abundant nuclear localization. In this RNP association, CBC facilitates mRNA maturation and its
nuclear export. Upon entry into the cytoplasm, CBC engages trRNP formation that facilitates the initial round(s) of
translation (2). This involves the direct association of CBP80 with either CTIF or eIF4G, which provides a molecular
bridge to eIF3 binding and 43S pre-initiation ribosome complex recruitment. This CBC trRNP also harbors nuclear
and cytoplasmic poly-A binding protein (PABP) at its 3' terminus and exon junction complexes (EJC) along the open
reading frame. These features are critical for the CBC trRNP in engaging translation activity by facilitating
interactions between the target transcript and the 43S pre-initiation ribosome complex. Remodeling of a transcript
from a CBC trRNP to an eIF4E trRNP follows the pioneer round(s) of translation and is critical for regulated steady-
state protein synthesis (3). This trRNP remodeling is facilitated in two ways. At the 5' terminus, exchange of CBC
for eIF4E is governed by a translation-independent mechanism whereby the strong affinity of CBP80 for the nuclear
import factor importin α1 (IMP α1) drives its association with the nuclear pore-associated karyopherin importin β1
(IMP β1). This CBC-IMP α1-IMP β1 complex formation results in CBC destabilization from the 5' cap and its
nuclear recycling. The association between CBC and IMP α1 is driven by the canonical nuclear localization
sequence (NLS) found within the N-terminus of CBP80. IMP α1 recruits IMP β1 via its IMP β1-binding domain
(IBB). Displacement of CBC from the 5' cap allows eIF4E to bind. eIF4E then draws stabilized associations
with eIF4G that recruit eIF4A and complete formation of the eIF4E trRNP at the 5' terminus. Removal of
exon junction complexes and the complete exchange of PABPN for PABPC occur in a translation-dependent
manner, although the exact molecular mechanisms remain unknown. Re-engagement of CBC in post-transcriptional
gene control is facilitated upon its nuclear re-entry and re-association with a nascent transcript (4). The maintained
nuclear interaction between CBC and IMP α1 is believed to prime for subsequent trRNP remodeling upon
The significance of the CBC trRNP, both in
composition and function, is for the mRNA
surveillance and quality control process of nonsense-
mediated mRNA decay (NMD) [79, 89]. NMD is
intricately linked to the pioneer rounds of translation
whereby it assesses initial mRNA integrity for
appropriate full-length protein production .
This activity involves the recognition and
resolution of premature termination events so as
to prevent production and accumulation of rogue
protein products . Critical interactions between
CBP80 and the NMD effector up-frameshift
protein 1 (UPF1) mediate this effect . The
associated exon junction complexes of the CBC
trRNP are also necessary for coordinating recognition
of premature termination events with subsequent
translation inhibition and directed mRNA decay
. Thus, the long-standing model has been that
the CBC trRNP is distinct for the pioneer round(s)
of translation in order to identify targets of NMD.
Recent studies, however, have provided significance
for the CBC trRNP beyond the pioneer round(s)
of translation and NMD. Such is the case for the
expression of antigenic peptides of the MHC class
I pathway . The ability to distinguish self
from non-self is critical to appropriate immune
function and recognition of invading pathogens.
The MHC class I pathway functions in both T-cell
education and activation of the immune system.
Critical to this effect is the generation of antigenic
peptides. Cap-dependent translation affords a
molecular basis for regulated peptide expression.
It was demonstrated that inhibition of eIF4E
trRNP dynamics impaired the production of full-
length protein products without consequence on
the generation of antigenic peptides .
Furthermore, the temporal regulation of antigenic
peptide production was shown to coincide with
prominent CBC trRNP activity . Thus, these
findings indicate a role for the CBC trRNP in the
innate immune response.
The CBC trRNP is also critical for the regulated
expression of the core histone proteins . Genome
integrity is dependent upon the effective packaging
of DNA into appropriate chromatin structure. This
effect is mediated by the coordination of histone
protein synthesis with DNA replication. Robust
histone protein production is observed during the
S phase of the cell cycle when DNA replication
The CBC trRNP
CBC is a heterodimeric protein complex of
two subunits: CBP80 and CBP20. CBP20 directly
binds to the 7-methyl guanosine cap of eukaryotic
mRNAs while CBP80 regulates this interaction
. The association of CBC with a 5' cap occurs
co-transcriptionally when a nascent transcript emerges
from the RNA polymerase II holoenzyme 
(Figure 7). This affinity is facilitated by the abundant
steady-state localization of CBC within the nucleus,
an effect driven by a bipartite nuclear localization
sequence within CBP80 and its association with
the nuclear import factor, importin-α1 [82, 83].
Here CBC is critical for the major post-transcriptional
events of 3' end processing and splicing that
mature an mRNA [80, 84, 85]. CBC then remains
bound to the 5' cap and facilitates mRNA nuclear
export [81, 83, 85-87].
Once in the cytoplasm, CBC engages the trRNP
activity that coordinates the so-called pioneer
round(s) of translation [88, 89] (Figure 7). These
encompass the initial interactions of a ribosome
with an mRNA that generate protein products.
This CBC trRNP is defined by CBC, bound to the
5' cap, and its direct association with the translation
initiation factor eIF4G or CTIF [90, 91]. A CBC-
eIF4G/CTIF interaction is important for generating a
molecular bridge with the translation initiation
factor eIF3, which recruits the 43S pre-initiation
ribosome complex to the 5' terminus and initiates
scanning [4, 90, 91].
Pioneer CBC trRNP complexes are also defined
by the presence of exon junction complexes and
the nuclear poly-A binding protein (PABPN)
[89, 92] (Figure 7). Exon junction complexes are
dynamic protein assemblies that organize upon the
coding sequence of an mRNA with the conclusion
of a splicing event and facilitate critical post-
transcriptional activities. One of these post-
transcriptional activities is to enhance the translation
of spliced mRNAs by providing a molecular bridge
with the 43S pre-initiation ribosome complex
. This occurs via a direct association between
the EJC interacting factor PYM and the 40S
ribosomal subunit . PABPN is a 3' associated
factor that is bound to the poly-A tail of mRNAs
and functions in the earlier post-transcriptional
event of 3' end processing .
124 Sarah Fritz & Kathleen Boris-Lawrie
presence of the eIF4E cap-binding protein bound
to the 5' cap of an mRNA in place of CBC. eIF4E
is selective for an association with eIF4G and
does not exhibit an interaction with CTIF, making
eIF4G another defining member of the eIF4E
trRNP . Furthermore, eIF4E selectively interacts
with the DEAD-box RNA helicase eIF4A such
that eIF4A is also a defining member of eIF4E
trRNP . The eIF4E trRNP is further distinguished
from the CBC trRNP by its absence of associated
exon junction complexes and its exclusive interaction
with the cytoplasmic poly-A binding protein (PABPC)
These characteristic differences between the eIF4E
trRNP and the CBC trRNP, both in composition
and temporal association with an mRNA, are
driven by the dynamic trRNP remodeling events
that occur following the pioneer round(s) of
translation (Figure 7). CBC has a high affinity for
the nuclear import factor importin α1, an association
that is driven by the classic nuclear localization
sequence in CBP80 . Notably, the interaction
of CBC with importin α1 is resistant to high salt
and observed throughout the nuclear-cytoplasmic
shuttling activity of CBC . Typical nuclear-
cytoplasmic shuttling proteins only demonstrate
a robust interaction with importin α1 in the
cytoplasm, as their binding is rapidly dissociated
upon nuclear import by the Ran-GTP gradient and
interactions with the nuclear export factor
exportin 2 (also known as CAS) . The
unique affinity of CBC for importin α1 primes
CBC for an interaction with importin β1 in the
cytoplasm . Importin β1 is the critical
karyopherin that drives importin α1-mediated
nuclear import by serving as a molecular bridge
between importin α1-cargo complexes and the
nuclear pore complex . A cytoplasmic CBC-
importin α1-importin β1 interaction destabilizes
CBC from the 5' cap, allowing eIF4E to bind and
for CBC to recycle to the nucleus [83, 99]. Impaired
importin α1-importin β1 dynamics reduces the
exchange of CBC for eIF4E on mRNAs .
Additionally, direct cofactor interactions can retain
CBC on target transcripts by functioning as a
molecular clasp that latches CBC onto the 5' cap
and prevents eIF4E association . Notably,
importin-driven CBC dissociation from the 5' cap
is critical for the molecular basis of trRNP remodeling
occurs. Completion of S phase triggers the rapid
degradation of histone mRNAs, which effectively
inhibits their expression and coordinates histone
production with DNA replication. The molecular
basis by which histone translation is linked to its
mRNA degradation is the CBC trRNP . Histone
mRNAs exhibit preferential association with the
CBC trRNP during steady-state translation due to
a direct interaction between CTIF and the
identifying 3' stem-loop binding protein (SLBP)
of histone mRNAs . This SLBP-CTIF interaction
generates a trRNP that facilitates efficient translation
of histone mRNAs during S phase and primes for
their rapid degradation upon the completion of
DNA synthesis . The rapid degradation of
histone mRNAs is driven by S phase-dependent
phosphorylation of UPF1 and its competition with
CTIF for SLBP association . The outcome is a
dynamic rearrangement in the CBC trRNP that
facilitates mRNA degradation . Collectively,
these findings indicate significance for the CBC
trRNP in the molecular basis of genome integrity.
An additional function for the CBC trRNP beyond
the pioneer round(s) of translation is cap-dependent
gene expression of HIV-1 . As previously
discussed, a hallmark feature of HIV-1 infection
is global suppression of host cell translation .
This effect is due to HIV-1-induced activation of
4E-BP1 and consequent suppression of eIF4E trRNP
activity . However, as an obligate parasite,
HIV-1 requires host cell translation machinery for
protein expression. Furthermore, it does so in an
arguably cap-dependent manner [33, 34]. This
seemingly paradoxical conundrum is resolved by
the virus selectively engaging the CBC trRNP for
expression of its critical structural proteins .
Unlike eIF4E, CBC is independent of regulation
by 4E-BP1 (see below). Furthermore, regulation
of CBC trRNP activity has yet to be identified
(see below). Therefore, this finding implies
significance for the CBC trRNP in maintained
cap-dependent translation during cell stress.
The eIF4E trRNP
The eIF4E trRNP dynamically assembles upon
an mRNA subsequent to the CBC trRNP and
facilitates steady-state protein synthesis [79, 99]
(Figure 7). The eIF4E trRNP is compositionally
distinguished from the CBC trRNP by the
RNA binding proteins target select mRNA templates for translation 125
4E-BP1-mediated translation control is considered.
As previously discussed, 4E-BP1 is a primary
target and effector of mTOR activation. mTOR is
the major cellular commander that translates
extracellular stimuli into coordinated effects on
protein synthesis. In an mTOR-induced hyper-
phosphorylated state, 4E-BP1 exhibits reduced
affinity for eIF4E [73-75]. This allows an
association between eIF4E and eIF4G that
facilitates a composed eIF4E trRNP to promote
steady-state translation [73-75]. These trRNP
dynamics are observed in response to mitogenic
stimuli [71, 76]. However, in cell stress, such as
amino acid deprivation or HIV-1 infection, mTOR
activation is reduced [71, 78]. This results in
hypophosphorylation of 4E-BP1 [71, 76, 78].
Hypophosphorylated 4E-BP1 exhibits a high
affinity for eIF4E that competes with eIF4G for
an association . The outcome is a 4E-BP1 :
eIF4E interaction that impairs eIF4E trRNP
complex formation and results in suppression of
eIF4E-mediated translation .
On the other hand, the CBC trRNP is insensitive
to regulation by 4E-BP1 [78, 102, 103]. This
results in its maintained translation activity during
cell stress events like hypoxia, serum starvation,
and HIV-1 infection, which evoke suppression of
mTOR activity and 4E-BP1 activation [78, 102,
103]. In fact, no regulators of cytoplasmic CBC
function have been identified. The nuclear activity
of CBC in pre-mRNA splicing is sensitive to
several extracellular stimuli such as growth factors
and UV irradiation. However the mechanisms
governing this effect and its significance for
downstream CBC trRNP function remain to be
elucidated . Likewise, CBC was identified as
a phosphorylation target of the ribosomal protein
S6 kinase (S6K) . Yet the actual occurrence of
CBC phosphorylation in cells and its significance
for regulating CBC trRNP activity remain ambiguous
A distinction between CBC trRNP regulation and
that of eIF4E trRNP control is significant for
eukaryotic translation in three main ways. First, it
affords the cell a mechanism to separate mRNA
surveillance translation activities from that of
steady-state protein synthesis. This allows for the
maintenance of critical quality control functions
in the advent of suppressed steady-state protein
synthesis. Second, it provides two distinct molecular
as eIF4E exhibits a lower affinity for 7-methyl-
guanosine relative to CBC . Thus, affinity
competition is not sufficient to drive eIF4E
association with the 5' cap even with eIF4E's
abundant cytoplasmic localization.
The binding of eIF4E to the 5' cap draws
stabilized associations with eIF4G that recruits
eIF4A and completes formation of the eIF4E trRNP
at the 5' terminus. Removal of exon junction
complexes and the exchange of PABPN for PABPC
occur with the pioneer rounds of translation .
The exact remodel mechanisms governing these
effects remain to be elucidated . It is important
to emphasize here a critical distinction between
trRNP remodeling at the 5' terminus from that at
the 3' terminus in the exchange of the CBC trRNP
for the eIF4E trRNP. As previously discussed,
exchange of CBC for eIF4E at the 5' cap is driven
by a translation-independent association of CBC
with the nuclear import factors importin α1 and
importin β1 . A CBC-importin α1-importin β1
complex formation destabilizes CBC from the 5'
cap, allowing eIF4E to bind . Remodeling at
the 3' terminus, however, with the removal of
exon junction complexes and the exchange of
PABPN for PABPC is dependent upon active
ribosome scanning and translation . These
mechanistic distinctions provide opportunities for
a transcript to undergo retention of CBC at the
5' cap but exhibit efficient remodel at the
3' terminus. In most instances, however, complete
trRNP exchange occurs and an mRNA becomes
fully engaged within the eIF4E trRNP to undergo
steady-state translation .
Regulation of the CBC and EIF4E trRNPs
Both the CBC trRNP and the eIF4E trRNP are
subjected to regulation by changes in the core
translation machinery. This includes phosphorylation
of eIF2α. CBC and eIF4E each interact with eIF2
and eIF3, implying similar associations with
the 43S pre-initiation ribosome complex .
Furthermore, introduction of a phosphomimetic
mutant of eIF2α significantly impairs nonsense
mediated mRNA decay to indicate that the
pioneer round of translation requires functional
eIF2α like steady-state protein synthesis .
A distinction is made between the regulation of
the CBC trRNP and that of the eIF4E trRNP when
126 Sarah Fritz & Kathleen Boris-Lawrie
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bases for cap-dependent translation control. This
feature is critical for enacting targeted protein
synthesis, especially during states of cellular
stress. Third, it presents an opportunity for
multiple integration mechanisms of invading viral
pathogens into the host translation process.
RNP biology is fundamental to eukaryotic protein
synthesis. Deregulated trRNP activity is associated
with cancer, neurological diseases and disorders,
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immune disorders [105-108]. Herein we provided
a comprehensive analysis of the trRNPs regulating
eukaryotic protein synthesis. We defined the core
trRNPs directing each stage of the translation
process and discussed their significance in facilitating
polypeptide production. Notably, we identified
distinct trRNPs with targeted activities in the
regulation of translation. These included the
specialized DExH/D-box RNA helicase trRNPs
and select RNA binding proteins that govern
protein synthesis at distinct stages of the translation
process on targeted mRNAs. Furthermore, we
discussed the molecular bases for the regulation of
trRNP activity, particularly the role of cell stress
in influencing trRNP dynamics and protein
production. Critically, we highlighted the differing
responses of each trRNP to cell stress and the
significance of this distinction for novel mechanisms
of translation control during the cellular stress
response. Collectively, our analyses demonstrated the
breadth and depth of trRNP biology in eukaryotic
protein synthesis. Future studies connecting trRNP
biology to the role of specialized ribosomes in
targeted translation control are interesting to consider.
We gratefully acknowledge all members of the
Boris-Lawrie lab for helpful discussion and Tim
Vojt for illustration and NIH P50CM103297.
CONFLICT OF INTEREST STATEMENT
The authors declare no competing financial interests.
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