WIREs RNA - 2022 - Naineni - Targeting DEAD Box RNA Helicases The Emergence of Molecular Staples

Received: 15 February 2022 Revised: 13 April 2022 Accepted: 16 April 2022
DOI: 10.1002/wrna.1738
ADVANCED REVIEW
Targeting DEAD-box RNA helicases: The emergence of

molecular staples
Sai Kiran Naineni1 | Francis Robert1 | Bhushan Nagar1 | Jerry Pelletier1,2,3
1
Department of Biochemistry, McGill
University, Montreal, Quebec, Canada
Abstract
2
Department of Oncology, McGill RNA helicases constitute a large family of proteins that play critical roles in
University, Montreal, Quebec, Canada mediating RNA function. They have been implicated in all facets of gene
3
Rosalind and Morris Goodman Cancer expression pathways involving RNA, from transcription to processing, trans-
Institute, McGill University, Montreal,
port and translation, and storage and decay. There is significant interest in
Quebec, Canada
developing small molecule inhibitors to RNA helicases as some family mem-
Correspondence bers have been documented to be dysregulated in neurological and neuro-
Jerry Pelletier, Room 810, 3655
Promenade Sir William Osler, Montreal,
development disorders, as well as in cancers. Although different functional
QC H3G 1Y6, Canada. properties of RNA helicases offer multiple opportunities for small molecule
Email: jerry.pelletier@mcgill.ca development, molecular staples have recently come to the forefront. These
Funding information bifunctional molecules interact with both protein and RNA components to
Canadian Institutes of Health Research, lock them together, thereby imparting novel gain-of-function properties to
Grant/Award Number: FDN-148366
their targets.
This article is categorized under:

Edited by: Purusharth Rajyaguru,
Associate Editor and Jeff Wilusz, Editor-
RNA Interactions with Proteins and Other Molecules > Small Molecule-
in-Chief RNA Interactions
RNA Interactions with Proteins and Other Molecules > Protein-RNA Inter-
actions: Functional Implications
KEYWORDS
chemical biology, DDX proteins, helicase inhibitors, molecular staples, RNA helicase
1 | INTRODUCTION
The life cycle of an RNA molecule is intricately linked to RNA helicases, a class of enzymes that reorganize RNA and
ribonucleoprotein (RNP) complexes by catalyzing unwinding and displacement reactions. Eukaryotic cellular RNA
helicases can be stratified based on their primary sequence motif composition and are assigned to two superfamilies
(SF), SF1 and SF2 (Jankowsky, 2011). The largest families are the DEAD-box and DEAH-box proteins, named based on
the canonical Asp-Glu-Ala-Asp/His sequence. Herein, we provide a brief description of the gene expression landscape
that DEAD-box family members have been implicated in and focus on small molecule discovery efforts directed toward
inhibiting their activity.
The RNA helicase fold shares a core composed of two RecA-like domains with conserved motifs that mediate ATP
binding, ATP hydrolysis, RNA binding, and RNA unwinding (Figure 1; Jankowsky, 2011). Helicases can adopt a wide
Sai Kiran Naineni and Francis Robert contributed equally to this study.
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2 of 22 NAINENI ET AL.
(a)
Ancillary Ancillary
N-term RecA-like Domain 1 RecA-like Domain 2 C-term
ATP Binding/Hydrolysis
Domain Domain
RNA Binding
Q I Ia Ib Ic II III IV IVa V Va VI
Linking ATP/RNA binding
(b)
4 Nuclear Processes
Cytoplasmic
Nuclear + Cytoplasmic
21
13
F I G U R E 1 RNA helicases are implicated in multiple gene expression regulatory steps. (a) DEAD-box RNA helicase domain
organization. Illustrated are the motifs conserved among family members and their implicated roles in ATP binding and hydrolysis, RNA
binding, and helicase activity. (b) Pie chart highlighting the number of helicases involved in gene expression pathways in the nucleus,
cytoplasm, or both. Data was compiled from Table S1
range of conformations to execute their function. The “open” conformation has the two RecA-like domains separated
and in different relative positions to each other. Upon binding ATP and RNA, extensive contacts occur between the two
RecA-like domains, stabilizing a “closed” conformation. ATP hydrolysis leads to RNA duplex unwinding and regener-
ates the open conformation (Andreou & Klostermeier, 2013). RNA helicases can also disrupt RNA–protein interactions
and serve as protein assembly platforms. They do not have stringent sequence specificity since most interactions with
RNA occur through backbone sugar–phosphate interactions (Jankowsky, 2011; Jarmoskaite & Russell, 2014; Leitao
et al., 2015; Ozgur et al., 2015; Putnam & Jankowsky, 2013). RNA selectivity as well as regulation of enzymatic activity
is generally imparted by auxiliary domains present at the extended amino and carboxy termini and/or protein co-factors
(Linder & Jankowsky, 2011). In this review, we restrict our discussion to DEAD-box helicase family members
(Fairman-Williams et al., 2010). There have been several excellent reviews on the roles of DEAD-box and DEAH-box
helicases in different biological processes and we refer the reader to these for more detailed insight (Bourgeois
et al., 2016; Putnam & Jankowsky, 2013).
2 | REGULATION OF GENE EXPRESSION BY RNA H ELICASES
RNA helicases have been implicated in all aspects of RNA biology and rather than attempting to provide an exhaustive
list of those involved in different gene regulatory pathways, we seek to provide here choice examples of processes in
which helicases have been implicated and to present a flavor of the types of regulation that have been documented. We
refer the reader to Table S1 for a list of DEAD-box proteins and gene expression pathways in which they have been
implicated.
2.1 | Transcription, splicing, and nucleo-cytoplasmic transport
In the nucleus, DEAD-box family members have been found to function as co-activators or co-repressors—interacting
with key components of the transcription apparatus to influence transcription (Fuller-Pace, 2006). For example, DDX5
is a transcriptional co-activator of the p53, vitamin D receptor, and androgen receptor genes (Bates et al., 2005; Clark
et al., 2008; Wagner et al., 2012), is over-expressed in many cancers, and has been implicated in promoting cell prolifer-
ation and epithelial-to-mesenchymal transitions (Yang et al., 2006). Depending on context, DDX5 and DDX17 can act
as transcriptional repressors by associating with histone deacetylase 1 (Wilson et al., 2004). DEAD-box helicases have
NAINENI ET AL. 3 of 22
also been found to impact the epigenome—for example, DDX18 has been implicated in binding polycomb repressive
complex 2, inhibiting rDNA methylation, and rRNA transcription (Zhang et al., 2020). Helicases are present in different
splicing complexes and some have been implicated in alternative splicing events (Bourgeois et al., 2016). For instance,
DDX48 has been implicated in regulating class-specific mRNA alternative splicing (Mazloomian et al., 2019) and
DDX41 mutations, associated with myeloid neoplasms, have been shown to lead to perturbations in exon skipping and
retention (Polprasert et al., 2015). Nucleo-cytoplasmic export of RNA is also dependent on RNA helicases. Indeed, the
first example of a metabolite regulating helicase activity emerged from studies assessing Dbp5's (DDX19B) role in remo-
deling RNPs at the nuclear pore complex (NPC; Alcazar-Roman et al., 2006; Weirich et al., 2006). Dbp5 is activated at
the NPC by its interaction partner, Gle1, and the Dbp5–Gle1 interaction is stabilized by inositol hexakisphosphate
(InsP6) which binds to the Dbp5–Gle1 interface (Montpetit et al., 2011). This is thought to fine-tune Dbp5–Gle1 interac-
tion strength and regulate mRNA export in response to different physiological stimuli. As well, DDX3X has been impli-
cated in the nuclear export of a number of viral RNAs and is being explored as an anti-viral drug target (Lai
et al., 2008).
2.2 | Exon–junction complex formation
The nonsense-mediated decay (NMD) surveillance pathway is dependent on DDX48, a critical component of the exon–
junction complex (EJC). The EJC is a multicomponent complex of four core proteins (DDX48, MAGOH, RBM8A/Y14,
CASC3/MLN51/BTZ) that forms upstream (20–24 nts) of exon–exon junctions and links mRNA export to translation
and degradation (Schlautmann & Gehring, 2020). During splicing, DDX48 adopts a closed conformation and binds ATP
when complexed with the splicing factor Cef1. The MAGOH-RBM8A heterodimer then binds DDX48, locking it in the
closed conformation by inhibiting DDX48 ATPase activity and stabilizing its RNA-bound state, thus preventing RNA
dissociation. This serves as a platform for the recruitment of auxiliary proteins. Disassembly of the EJC leads to ATPase
activation and RNA disengagement as DDX48 transitions to an open conformation (Andersen et al., 2006; Ballut
et al., 2005; Bono et al., 2006). The role of DDX48 in EJC formation thus provides a paradigm for how regulating heli-
case conformational transitions can extend the function of this class of proteins to where they serve as scaffolds for
multi-component complex assembly. The EJC is essential for triggering NMD when a premature termination codon
(PTC) is present upstream (>50–55 nts) of the EJC, a process that serves to protect cells from producing truncated pro-
tein products that could exhibit harmful gain-of-function or dominant-negative activity (Figure 2; Kurosaki
et al., 2019).
2.3 | Ribosome RNA processing
The synthesis and maturation of ribosomes is another nuclear process that is highly dependent on the activity of RNA
helicases. Three (28S, 18S, and 5.8S) of the four ribosomal RNAs (rRNAs) are derived from a common RNA Polymerase
I precursor, whereas the 5S rRNA is transcribed by RNA Pol III. More than 200 factors and 75 small nucleolar RNAs
(snRNAs) have been implicated in ribosome synthesis in yeast, among which are 15 DEAD-box RNA helicases (Martin
et al., 2013). Studies in human cells have lagged behind those of yeast, but 22 DEAD-box RNA helicases have been
implicated in mammalian ribosomal production and biogenesis (Figure 2 and Table S1).
2.4 | Translation
The best-characterized helicase, and a founding member of the DEAD-box family, is eIF4A (DDX2)—a factor essential
for recruiting ribosomes to most mRNA templates. Ribosome recruitment occurs by either a cap-dependent or cap-
independent mechanism; the latter involving internal recruitment of ribosomes within mRNA 50 leader regions
(Pelletier & Sonenberg, 2019). Most eukaryotic cellular translation occurs via a cap-dependent process and involves
eIF4F—a complex consisting of (i) eIF4E, the cap-binding protein responsible for interacting with mRNA cap struc-
tures; (ii) eIF4A1 (DDX2A) or eIF4A2 (DDX2B), RNA helicases that facilitate access of the 43S ribosomal complex to
mRNAs; and (iii) eIF4G, a scaffold protein that mediates mRNA binding to the 43S pre-initiation complex (PIC;
Pelletier & Sonenberg, 2019). eIF4A1 and eIF4A2 share 90% amino acid identity and are functionally interchangeable
FIGURE 2 Schematic diagram implicating DEAD-box helicases in mammalian rRNA production and biogenesis, NMD, and translation
initiation
(Conroy et al., 1990; Yoder-Hill et al., 1993). eIF4A1 is the more abundantly expressed homolog and is essential,
whereas eIF4A2 is not (Senechal et al., 2021).
Another helicase implicated in translation is Ded1 (DDX3X; Chuang et al., 1997; de la Cruz et al., 1997). In yeast,
eIF4A is required for translation of all mRNAs regardless of their 50 leader complexity whereas Ded1 is dedicated to
resolving structural barriers to promote ribosome recruitment (Guenther et al., 2018; Gupta et al., 2018; Sen et al., 2015;
Yourik et al., 2017). When Ded1 is rendered limiting, structure in the mRNA 50 leader region is stabilized, leading to
increased initiation at upstream near-cognate start codons. This is coupled to a concomitant reduction in translation
initiation from downstream major open reading frames (ORFs; Guenther et al., 2018). In mammalian cells, DDX3X has
been found to associate with the 40S ribosome near the mRNA entry site and its knockdown preferentially leads to
reduced translation of mRNAs with highly structured 50 leader regions (Figure 2; Calviello et al., 2021).
2.5 | Messenger RNA degradation
Messenger RNA degradation also involves the intimate participation of RNA helicases. DDX5 has been reported to
enhance NMD (Geissler et al., 2013), yeast Dhh1 (DDX6) stimulates decapping and mRNA degradation (Coller
et al., 2001; Fischer & Weis, 2002), and DDX48 is required for NMD-mediated elimination of PTC containing mRNAs.
2.6 | MicroRNA-mediated silencing
RNA helicases have been implicated in different steps of microRNA-mediated silencing (Table S1). DDX5, DDX17,
DDX1, DDX23, and DDX25 are involved in the maturation of specific pri-miRNAs (Bourgeois et al., 2016). DDX5 and
DDX20 have been implicated in miRNA-induced silencing complex (RISC) formation (Bourgeois et al., 2016). In mam-
mals, DDX6 plays a role in the inhibition of translation by microRNAs, where it interacts with the RISC. Through a
series of protein interactions, DDX6 fosters recruitment of the cap-binding protein 4EHP to the mRNA 30 -UTR (Chapat
et al., 2017). 4EHP does not interact with eIF4G so upon cap-binding by 4EHP, the mRNA is held in a circular configu-
ration, and ribosome recruitment is inhibited through occlusion of eIF4F:cap interaction.
2.7 | Cytoplasmic transport and storage
The transport of mRNA and its storage in RNA granules is an important aspect of gene regulation that impacts transla-
tion and decay. Appropriate localization of mRNAs within cells ensures that some protein products are produced only
where required through localized translation (Buxbaum et al., 2015). To this end, several helicases have been found to
participate in RNA transport or to be required for localized translation (Bourgeois et al., 2016). RNA storage granules
provide safe harbors where mRNAs can be temporarily docked in response to stress or specific developmental events,
and these are also known to contain, and their presence influenced by, DEAD-box family members (Hilliker, 2012).
DDX19A and eIF4A1 have been shown to inhibit RNA condensation and prevent the formation of stress granules,
implying a role as RNA chaperones (Tauber et al., 2020).
The few examples provided above serve to highlight the extent to which RNA helicases influence gene expres-
sion and the central role they play in RNA biology. The finding that most helicases can participate in more than
one step of gene expression implies a layer of coordination between different processes that is yet to be fully appre-
ciated. For example, of the 38 DEAD-box helicases, 21 are implicated in solely nuclear, and four regulate only
cytoplasmic, events (Figure 1b). Thirteen helicases are thought to be involved in both nuclear and cytoplasmic
events. An open question is whether these afford cross-regulation of gene expression between these cellular com-
partments. How the activity of a given DEAD-box protein affects the connectivity between different gene regula-
tory pathways will provide insight into systems-level type of expression regulation and represents an important
future challenge to explore.
3 | R N A HE L I C A S E S A S P O T E N T I A L T A R G E T S I N C A N C E R
Deregulated helicase activity has been implicated in tumor initiation, maintenance, and drug response and this war-
rants their exploration as anti-neoplastic targets. Along these lines, several DEAD-box family members (i) are over-
expressed in human cancers (Abdelhaleem, 2004; Robert & Pelletier, 2013), (ii) can impact the expression of prominent
oncogenes or oncogenic pathways (Bates et al., 2005), and (iii) are partners of oncogenic fusions in cancer (e.g., NUP98-
DDX10, DDX6-FOXR1; Picco et al., 2019; Yassin et al., 2010). There are three cellular processes involving RNA
helicases where there is a preponderance of evidence supporting the exploration of DEAD-box proteins as anti-cancer
targets.
3.1 | Translation initiation
Ribosome recruitment is the rate-limiting step of translation and eIF4E is the least abundant of all translation factors
rendering this step a critical checkpoint for regulation (Pelletier & Sonenberg, 2019). In addition, mRNAs show differ-
ential dependencies on the eIF4F complex for initiation and secondary structure within the mRNA 50 leader is one fea-
ture that dictates this—increased structure is associated with reduced competition of that mRNA for eIF4F and lowered
translational output (Graff et al., 2008). In tumor cells, two potent signaling pathways impinge on eIF4F to increase
activity and this leads to selective stimulation of translation initiation—with some of the responsive mRNAs encoding
oncogenic effectors. First, assembly of the eIF4F complex is controlled by the PI3K/Akt/mTORC1 signaling pathway,
where its activation leads to phosphorylation of the eIF4E binding protein, 4E-BP1, reducing its association with eIF4E
and increasing eIF4F complex formation. As well, an inhibitor of eIF4A, PDCD4, is under regulation of the PI3K/Akt/
mTORC1 axis (Dorrello et al., 2006; Yang et al., 2003). Hyperactivation of mTORC1 leads to S6Kinase-mediated phos-
phorylation of PDCD4 and subsequent degradation of PDCD4—impacting translation initiation (Dorrello et al., 2006).
A pan-cancer proteogenomic analysis of 11,219 cancers from 32 major types revealed that the PI3K-mTORC1 signaling
pathway is hyper-activated in >70% of cancers (Zhang et al., 2017). Second, analysis of the RAS–ERK pathway in 9125
samples from 33 cancer types in TCGA indicated that this pathway is mutated in 46% of cancers analyzed (Sanchez-
Vega et al., 2018). RAS–ERK activation leads to MNK1/2-mediated phosphorylation of eIF4E at S209, an event that
increases eIF4F activity (Pelletier & Sonenberg, 2019). Hence, targeting eIF4F has emerged as an approach by which to
inhibit tumor cell proliferation (Bhat et al., 2015).
Other helicases have also been implicated in translation initiation, but their roles are not as well defined as for
eIF4A. DDX3X (Ded1) plays a critical role in 43S ribosome scanning in yeast (see above). In mammalian initiation,
depending on the experimental context, DDX3X has exhibited stimulatory or inhibitory activity towards translation
(reviewed in [Parsyan et al., 2011]). Recent experiments based on ribosome profiling and PAR-CLIP experiments have
invoked a stimulatory role in the translation of mRNAs with structured 50 UTRs (Calviello et al., 2021). DDX3X and its
homolog DDX3Y can functionally complement each other in translation in human cells (Venkataramanan et al., 2021).
The Drosophila melanogaster DDX4 homolog, Vasa, is essential for embryonic patterning and germ cell specification. It
is recruited to the 30 UTR of specific mRNAs and interacts with eIF5B, a factor required for joining of the 60S and 40S
subunits (Parsyan et al., 2011). Consistent with a similar mechanism at play for DDX4 in mammalian cells, mapping of
DDX4 binding sites on mRNAs revealed predominant interactions around start codons, stop codons, and 30 UTRs in
RNA from mouse oocytes (Su et al., 2021). Dbp5 (DDX19B) has been implicated in translation termination where it
interacts with eukaryotic release factor 1 (Gross et al., 2007). It also ensures that the first round of translation to occur
following nuclear exit of the mRNA is restricted to the perinuclear region where DDX19B is localized (Park
et al., 2021). DDX25 is required for spermatogenesis and has been implicated in the regulation of specific mRNAs
required for this developmental process. Although a mechanism is lacking, it appears that stimulation of translation by
phospho-DDX25 is 30 UTR-dependent (Kavarthapu et al., 2019). DDX41 has been assigned a role in repressing the
expression of the cyclin-dependent kinase inhibitor p21 through binding to the 30 UTR of p21 mRNA (Peters
et al., 2017). The effect appears to be p53-dependent and potentially places DDX41 within the p53 regulatory node.
3.2 | Nonsense-mediated decay
The process of NMD has also been implicated in cancer initiation, especially when PTCs arise in tumor suppressor
genes (Huusko et al., 2004; Ionov et al., 2004; Lindeboom et al., 2016). A meta-analysis of over 1 million mutations
across 24 cancer types in TCGA identified >70,000 NMD-eliciting mutations (6%) with the gene most widely affected
by NMD being TP53 (Hu et al., 2017). Inhibition of NMD and PTC readthrough is thus being explored in cancer biology
as a way of reactivating tumor suppressor gene expression, as well as increasing tumor cell visibility to the immune sys-
tem by enabling the production of unique C-terminal peptides downstream of frame-shift mutations. Suppression of
NMD is thus an approach that would allow tumor cells to become re-sensitized to chemotherapeutic agents and
increase anti-tumor immunogenicity (Litchfield et al., 2020; Pastor et al., 2010; Popp & Maquat, 2015).
3.3 | Ribosomal RNA biogenesis
Regulation of ribosome biogenesis is fundamental to normal cell growth and proliferation and its acceleration is associ-
ated with malignant transformation. Increased rRNA synthesis is augmented in the presence of elevated AKT/mTORC1
signaling and increased MYC levels (Chan et al., 2011; Hsieh et al., 2015). On the other hand, inhibition of rRNA
processing induces cell cycle arrest that is p53-RB1 dependent (Bhat et al., 2004; Dai et al., 2004; Fumagalli et al., 2009;
Lohrum et al., 2003; Pestov et al., 2001; Rubbi & Milner, 2003; Sulic et al., 2005). This is a consequence of increased
pools of several ribosomal proteins that sequester MDM2/HDM2 from TP53, leading to TP53 stabilization. TP53 then
induces p21 expression, which in turn blocks RB1 phosphorylation and inhibits the activity of E2F transcription regula-
tors to prevent cell cycle progression (David-Pfeuty, 2006; Sherr & Roberts, 1999). This checkpoint serves to prevent
cells from dividing before a threshold level of ribosomes is available to enable daughter cell survival. In cancer cells, dis-
ruption of RB1 or p53 function leads to severance of the ribosome biogenesis and cell cycle link. Hence, even in the face
of rRNA synthesis inhibition in cancer cells, cell division proceeds despite reduced ribosome content; leading to a situa-
tion that is incompatible with the survival of daughter cells (Montanaro et al., 2007). As such, blocking RNA Pol I activ-
ity with small molecule inhibitors (e.g., CX-3543, CX-5461, BMH-21) is being explored as an anti-cancer approach
(Ferreira et al., 2020; Heerma van Voss et al., 2018). It remains an open question if inhibition of rRNA processing will
phenocopy RNA Pol I inhibition.
4 | SMALL M OLECU LE IN H I B IT O R S O F DE A D- B O X R NA H EL I C A SE S
RNA helicases offer multiple opportunities for drug development (Figure 3). Small molecules that block protein:heli-
case interaction, interact with the ATP binding pocket, nucleic acid interaction interface, or inhibit ATPase hydrolysis
have been described (Figure 3a). The RecA-like domains of DEAD-box proteins are connected by a flexible linker and
their opening and closing is essential to helicase function with each pose having the potential to accommodate different
small molecules (Figure 3b). Finally, compounds that “staple” a DEAD-box protein onto RNA have also been described
(Figure 3c). Currently, there are only a small number of RNA helicases against which small molecule inhibitors have
been identified and we provide a brief summary of their development status.
4.1 | DDX2A/DDX2B (eIF4A1/eIF4A2)
The first selective, and among the most potent RNA helicase inhibitors identified, were three natural products that
emerged from a high throughput screen (HTS) in search of translation initiation inhibitors. These compounds were ulti-
mately found to target eIF4A1 and eIF4A2. Hippuristanol binds the C-terminal domain of eIF4A and interferes with
RNA binding by stabilizing eIF4A in the closed conformation (Figure 3b; Sun et al., 2014). It interacts with amino acids
within, but also adjacent to, C-terminally located motifs Va and VI (Figure 1a) (Lindqvist, Oberer, et al., 2008). The
adjacent residues are not conserved among other DEAD-box family members and this rationalizes the selectivity of
hippuristanol for eIF4A (Lindqvist, Oberer, et al., 2008). Hippuristanol was shown to also inhibit DDX48 RNA-
dependent ATPase activity in vitro, although the IC50 for inhibition was 10-fold higher than for eIF4A1 (Lindqvist,
Oberer, et al., 2008), and this lower response was attributed to several amino acid substitutions within the DDX48
hippuristanol binding site. A CRISPR/Cas9-based variomics screen in Hap1 cells, using a collection of sgRNAs tiling
the eIF4A1 coding region, identified several hippuristanol-resistant alleles; and these were instrumental in genetically
linking the translation inhibitory and cytotoxicity properties of hippuristanol to eIF4A1 target engagement (Steinberger
et al., 2020). Genome-wide translation profiling uncovered that length, overall secondary structure, and cytosine con-
tent of the 50 leader region are defining features of hippuristanol-responsive mRNAs (Steinberger et al., 2020).
Hippuristanol has shown effectiveness as a single agent, or in combination with several other chemotherapeutics, in
preclinical models (Chu & Pelletier, 2015; Shen & Pelletier, 2020). The two other compounds that emerged from the
aforementioned HTS were rocaglates and pateamine A (PatA and analogs), and will be elaborated on further below.
A number of additional compounds have been proposed to inhibit eIF4A1 (Figure 3). These include elatol and
elisabatin A which were found to inhibit ATP hydrolysis (Peters et al., 2018; Tillotson et al., 2017). The prostaglandin
15d-PGJ2, has been reported to bind to eIF4A1 and inhibit eIF4A–eIF4G interaction (Kim et al., 2007; Yun et al., 2018).
Also, both 6-aminocholestanol (6-AC) and sanguinarine (SAN) were reported to block eIF4A ATPase and helicase
F I G U R E 3 Schematic outline of various activities of DEAD-box helicases that can be targeted for small molecule inhibition. Shown are
known helicase inhibitors, the step that they interfere with, and their molecular targets (in parenthesis). (a) Inhibitors can target interacting
proteins, ATP/RNA binding, RNA unwinding, or translocation along with the mRNA template. (b) Hippuristanol is a natural product that
traps eIF4A in the closed conformation, preventing it from interacting with RNA (Sun et al., 2014). (c) PatA (and analogs) and rocaglates act
as molecular staples to trap eIF4A onto RNA. + indicates secondary targets with reduced affinity or activity
activity (Abdelkrim et al., 2018; Harigua-Souiai et al., 2018; Jiang et al., 2019). Recently, a substituted quinoline analog,
cmpd 28, was identified following a screen for inhibitors of eIF4A ATPase activity and found to be an RNA-competitive,
ATP-uncompetitive inhibitor (Zerio et al., 2021). We await experiments addressing the selectivity of these compounds
in order to attribute any reported biological activity to eIF4A inhibition. It is concerning to us that exposure of cells to
several of these compounds (elatol, SAN, 15d-PGJ2) induced eIF2α phosphorylation—a potent, eIF4A-independent
mechanism that will lead to inhibition of translation (Naineni et al., 2020).
4.2 | DDX3X/DDX3Y
The human DDX3 paralogs are located on the X (DDX3X) and Y (DDX3Y) chromosomes, and the two encoded proteins
show 92% amino acid identity. DDX3X is a multifaceted protein that has been implicated in transcription, pre-mRNA
splicing, RNA export from the nucleus, translation, and microRNA expression (Mo et al., 2021; Table S1). Depending
on context, DDX3X has been ascribed to harbor tumor suppressor or oncogenic activity (Bol, Xie, & Raman, 2015; Mo
et al., 2021). Mutations in DDX3X have been reported in head and neck tumors (4%; Stransky et al., 2011), chronic mye-
loid leukemias (3%; Wang et al., 2011), melanomas (6.4%; Alkallas et al., 2020; Vanni et al., 2020), and
medulloblastomas (8–10%; Jones et al., 2012; Pugh et al., 2012; Robinson et al., 2012). As well, germline mutations in
DDX3X have been associated with intellectual disability (Snijders Blok et al., 2015). There has thus been significant
interest in identifying small molecules by which to probe DDX3X activity in various biological contexts. As well,
DDX3X has been implicated as an essential co-factor for several viruses, and inhibitors are being sought as anti-viral
agents (Maga et al., 2011).
Several types of molecules have been developed that target the ATPase activity of DDX3X—rhodamine analogs
(FE15, FE109; Maga et al., 2008) [which were later converted to triazine scaffolds (FE87; Maga et al., 2011)] and ring-
expanded nucleosides (RENs; Figure 3a; Yedavalli et al., 2008). FE87 showed low micromolar inhibition of HIV replica-
tion in infected peripheral blood mononucleated cells (PBMCs) (Maga et al., 2011). The best characterized RENs
are RK33 (Bol, Vesuna, et al., 2015; Kondaskar et al., 2010) and NZ51 (Xie et al., 2015). RK33 has been shown to bind
DDX3X, but not DDX5 or DDX17 (Bol, Vesuna, et al., 2015). These compounds exert anti-proliferative activity toward
breast cancer (IC50, 2–10 μM; Xie et al., 2015), lung cancer (IC50, 4–8 μM; Bol, Vesuna, et al., 2015), medulloblastoma
(IC50, 2.5–3.5 μM; Tantravedi et al., 2019), and Ewing sarcoma cell lines (IC50, 5 μM; Wilky et al., 2016). RK33 can
induce radiosensitivity in orthotopic lung (Bol, Vesuna, et al., 2015) and prostate (Xie et al., 2016) cancer models. At
low micromolar concentrations, RK33 also shows broad anti-viral activity (Yang et al., 2020). Toxicology, bio-
distribution, pharmacokinetic analysis, and metabolism of RK33 showed it to be non-toxic in mice (Bol, Vesuna,
et al., 2015). Experiments with RK33 in combination with radiation-induced tumor regression in a lung cancer xeno-
graft model in vivo (Bol, Vesuna, et al., 2015).
Virtual docking approaches followed by chemistry optimization led to the identification of E101D, a compound that
blocked DDX3X ATPase and helicase activity, while showing no activity toward DDX1 or the HCV NS3 helicase (Radi
et al., 2012; Figure 3a). Improvement on this molecule led to the discovery of cmpd 16d which was found to inhibit
HIV (IC50, 1 μM), HCV (IC50, 1 μM), DENV (IC50, 2.6 μM), and WNV (16.5 μM) replication (Brai et al., 2016). A third-
generation series of related structures yielded several that inhibited DDX3X helicase activity and exhibited anti-HIV
(Brai, Riva, et al., 2020) and anti-DENV-2 (Brai, Boccuto, et al., 2020) activity. Further optimizations lead to the devel-
opment of BA103, a molecule that was well tolerated in mice and reduced tumor growth in vivo in a xenograft model of
glioblastoma (Brai et al., 2021).
Other inhibitors of DDX3X that have been identified include (i) Kerolac salt which inhibits ATPase activity and was
shown to suppress oral carcinogenesis in vivo (Samal et al., 2015), (ii) compounds that, through molecular docking were
predicted to bind to a unique pocket in DDX3X and subsequently found to inhibit helicase activity (Riva et al., 2020),
and (iii) cmpd C1 that emerged following an HTS campaign and inhibited DDX3X helicase activity [but not eIF4A3
(DDX48)] (Nakao et al., 2020; Figure 3).
A critical piece of information that is lacking among studies on DDX3X inhibitors is experiments demonstrating
that the biological activities observed with these compounds are due to on-target engagement. For example, depletion
of DDX3X has been shown to lead to repression of translation of mRNAs with complex 50 -UTRs, but RK-33 treatment
of cells does not recapitulate these results (Calviello et al., 2021). Rather, RK-33 appears to be a general inhibitor of
translation [see figure S5C in (Calviello et al., 2021)]. There is thus an urgent need for DDX3X inhibitors that have been
rigorously shown to exert on-target biological activities.
4.3 | DDX5
DDX5 is another multifunctional helicase with roles as a transcriptional co-factor, in mRNA and microRNA
processing, ribosome biogenesis, and RNA turnover (Xing et al., 2019). It activates signal transduction pathways
(Wnt, Notch, and mTOR) and elevated levels have been reported in tumor cells (Xing et al., 2019). Upon phos-
phorylation, DDX5 is translocated to the cytoplasm where it facilitates the cytoplasmic accumulation of β-catenin
leading to activation of Wnt target genes, such as cyclin D1 and Myc (Xing et al., 2019). Hence, there is significant
interest in targeting DDX5 in cancer. RX-5902 is a compound found to bind phospho-DDX5 and inhibit β-catenin
dependent ATPase activity while having no impact on DDX5 RNA-dependent ATPase activity (Kost et al., 2015).
Treatment of cancer cells with RX-5902 lead to downregulation of Bcl-2 and several proliferation-associated genes,
resulting in a G2/M cell cycle blockade and triggering of cell death (Capasso et al., 2019). RX-5902 has been inves-
tigated for its antiproliferative activity toward breast cancer cells as a single agent, as well as its ability to enhance
immunotherapy (Tentler et al., 2020). RX-5902 has been tested in a phase I clinical trial and the results indicate it
to be well tolerated (Diamond et al., 2017).
4.4 | DDX39B
DDX39B has been implicated in the assembly of a large, viral ribonucleoprotein particle (hTREX) required for herpesvi-
rus replication (Schumann et al., 2016). A virtual docking screen identified CCT018159 as an ATP/ADP competitive
inhibitor of DDX39B capable of disrupting TREX formation (Schumann et al., 2016). CCT018159 was shown to block
replication of HSV-1 and HCMV, as well as of the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)
(Schumann et al., 2016). CCT018159 is also an inhibitor of HSP90 (Dymock et al., 2005), but its activity toward herpes
virus replication appears distinct from effects on HSP90 since the HSP90 inhibitor 17-DMAG (which does not target
DDX39B) failed to block KSHV replication.
4.5 | DDX41
DDX41 has been implicated in the processing and maturation of small nucleolar RNAs (snoRNAs) and its suppression
leads to rRNA processing defects due to inadequate rRNA pseudouridylation (Chlon et al., 2021). DDX41 also senses
cytoplasmic dsDNA and induces innate immune signaling through STING (Zhang et al., 2011). Germline mutations in
DDX41 are associated with a myelodysplastic syndrome which can transform into secondary leukemias (Sperling
et al., 2017). One prevalent DDX41 mutation is R525H, which maps to the C-terminal conserved motif (VI) and has
been implicated in ATP binding and hydrolysis (Polprasert et al., 2015). Hence, there is interest in finding small mole-
cules that inhibit the activity of the DDX41(R525H) mutant protein. To this end, a high throughput screen of 500,000
compounds against wild-type DDX41 and DDX41(R525H) has been performed in search of ATPase inhibitors. From
this screen emerged 26 primary hits, the biological activity of which has yet to be reported (Yoneyama-Hirozane
et al., 2017).
4.6 | DDX48
The desire to inhibit NMD in cancer has led to the identification and development of a series of DDX48 inhibitors. The
first compounds developed were indole-2-carboxylic acid derivatives found to be competitive inhibitors of ATP binding
(Ito, Iwatani, et al., 2017). These blocked RNA-dependent ATPase activity at an IC50 1 μM (Ito, Iwatani, et al., 2017)
with no activity (IC50 > 100 μM) toward eIF4A1, eIF4A2, DHX29, and BRR2 (Ito, Iwatani, et al., 2017). A lead com-
pound from this series inhibited DDX48 helicase activity but was ineffective at blocking NMD due to low cellular per-
meability (Ito, Tanaka, et al., 2017).
A second chemical series was more extensively explored. These were 1,4-diacylpiperazine derivatives, exemplified
by compound 52a (Figure 3a), which were found to inhibit DDX48 ATPase activity (IC50 0.2 μM; Ito, Tanaka,
et al., 2017). These compounds bind to the C-terminal domain of DDX48 and inhibit ATPase activity through an alloste-
ric mechanism (Iwatani-Yoshihara et al., 2017). Optimization for in vivo studies led to the development of compound
1q, which was shown to block NMD and reduce HCT-116 colorectal cancer cell growth in a xenograft setting (Mizojiri
et al., 2017). Compound 1q did not inhibit (IC50 > 100 μM) eIF4A1, eIF4A2, DHX29, or BRR2 (Mizojiri et al., 2017). A
CRISPR/Cas9-based variomics screen was used to identify and characterize DDX48 alleles resistant to growth inhibition
by compounds 52a and 1q, thus providing genetic evidence that NMD inhibition by these molecules is linked to DDX48
targeting (Cencic et al., 2020).
5 | M O L E C U L A R ST A P L E S TA R G E T T I N G E I F 4 A
The rocaglates and pateamines (Figure 4a,b) function by clamping eIF4A to RNA and preventing the dynamic motions
required for helicase function. As both compounds target a macromolecular complex, they can be classified as interfa-
cial inhibitors or molecular staples (Pommier & Marchand, 2011). Examples of drugs that act as interfacial inhibitors
and stabilize protein:protein (e.g., rapamycin and FKBP-mTOR, cyclosporin, and cyclophilin–calcineurin) or protein:
DNA (e.g., campothecin and topoisomerase I-DNA) complexes have been previously described (Pommier &
Marchand, 2011). As far as we are aware, rocaglates and pateamines represent the first example of small molecules that
“staple” protein:RNA complexes.
F I G U R E 4 Structural basis for the interactions between eIF4A1 and RocA and eIF4A1 and DMPatA. (a) Chemical structure of RocA.
(b) Chemical structure of DMPatA. (c) Structure of RocA bound to eIF4A1•poly (AG)5•AMPPNP (PDB 5ZC9). The close-up view to the right
indicates the interactions between RocA, eIF4A1, and RNA. The NTD and CTD are represented as gray and blue surface models,
respectively. The RNA is colored pink. Polar interactions are shown as red dashed lines, while non-polar interactions are shown as yellow
dashed lines. (d) Structure of DMPatA bound to eIF4A1•poly (AG)5•AMPPNP (PDB 6XKI). The close-up view to the right denotes the
interactions between DMPatA, eIF4A1, and RNA. (e) Overlay of eIF4A1-bound DMPatA (green) and RocA (orange) shown in two views.
Pink spheres represent a modeled uracil base at the position of the A7 RNA base. The transparent gray spheres on the right indicate the
additional volume of A7 from the structure. The model predicts weaker interactions between RocA and RNA when uracil replaces A7,
whereas stacking interactions with the DMPatA conjugated trienyl arm are maintained
5.1 | Molecular basis for eIF4A:RNA targeting
The biological activity of rocaglates was linked to eIF4A inhibition through mutagenesis studies in yeast, where several
TIF1 (yeast eIF4A) missense mutations were identified as rocaglate-resistant alleles and these mapped to the RNA bind-
ing interface (Sadlish et al., 2013). The engineering of one of these mutations, F163L, into the eIF4A1 alleles in NIH3T3
cells, generated a rocaglate-resistant cell line that was critical in linking the anticancer activity of these compounds to
eIF4A1 inhibition (Chu et al., 2016).
The crystal structure of RocA bound to eIF4A•poly(AG)5•AMPPNP provided welcomed insight into the mechanism
of clamping by this class of molecules (Iwasaki et al., 2019). RocA interacts with eIF4A through π–π stacking interac-
tions between two aryl rings (rings B and C) and F163 as well as hydrogen bonding between Q195 and the carbonyl
group of the 2-N,N-dimethyl-carboxamide (Figure 4c). Interactions with adjacent AG bases occur via hydrogen bonding
between the C8b hydroxyl group and N7 of the guanine base as well as stacking interactions between rings A and
B. RNA base A7 stacks with ring A of RocA. The crystal structure clearly provides molecular understanding of why the
eIF4A1 F163L allele is resistant to rocaglates (Chu et al., 2016). RocA has also been shown to induce clamping of
DDX3X to RNA, albeit with a 30-fold lower affinity than for eIF4A1 to RNA (Chen et al., 2021). This lower affinity can
be rationalized since DDX3X has a valine substitution at the critical F163 location.
The crystal structure of the PatA analog, DMPatA, in complex with eIF4A1•poly(AG)5•AMPPNP, revealed that the
same pocket targeted by RocA is also occupied by DMPatA (Figure 4d). Remarkably, the same amino acids critical for
rocaglate interaction (F163, Q195) are also employed by DMPatA (Figure 4d, right panel). F163 interacts with DMPatA
through bifurcating interactions with the thiazole ring and the E,Z-dienoate. Q195 forms van der Waals interactions
with the thiazole ring and D198 forms a salt bridge with the C3-primary amine. The tertiary amine at the end of the
trienyl arm makes weak interactions with R282 and D305 located in the eIF4A1 C-terminus. The E,Z-dienoate π-stacks
with G8 of the RNA segment and the conjugated trienyl arm π-stacks with an adjacent adenine base (A7) on one side
and the edge of G8 on the other side, while also making contact with R110.
PatA and the analog, DMDA-PatA, have also been shown to interact with DDX48 and inhibit NMD (Dang
et al., 2009; Figure 3c). PatA was found to stabilize the interaction of the EJC with RNA through its interaction with
DDX48. Coincident with this stabilization was perturbation of the interaction of Upf1 with the EJC which led to NMD
inhibition (Dang et al., 2009). The structural basis for this extended targeting profile is now understood since the critical
amino acids in eIF4A responsible for mediating interactions with PatA analogs are also present in DDX48 (F168, Q200;
Naineni et al., 2021).
Overlay of the eIF4A1-bound RocA and DMPatA molecules showed remarkable similarity in shape and bond
angles, despite the two compounds having unrelated chemical structures (Figure 4e, left). A key difference between
RocA- and DMPatA-induced clamping is RNA selectivity. RocA cannot accommodate binding of polypyrimidine-
containing RNA sequences whereas DMPatA shows no such discrimination (Naineni et al., 2021). Substitution of
purine bases with pyrimidines in the RNA of the RocA structure is predicted to lead to weakened interactions due to
loss of hydrogen bonding of the C8b-hydroxyl group to the RNA base as well as weaker stacking interactions between
the pyrimidine base and RocA ring A (Figure 4e). However, in the DMPatA structure, the extended trienyl amine side
chain maintains stacking interactions with pyrimidine bases (Naineni et al., 2021). This may explain the increased
potency of PatA analogs, relative to rocaglates, and although not yet demonstrated, it is expected that PatA analogs will
induce clamping to a broader set of mRNAs than rocaglates.
5.2 | Mechanism of translation inhibition
Stabilization of eIF4A:RNA complexes imparts a gain-of-function to eIF4A. Both rocaglates and pateamines stabilize
the closed eIF4A conformation on RNA resulting in long-lived complexes. Whereas the dissociation of eIF4A1 from
RNA occurs rapidly in vitro (on the order of 1–3 mins), in the presence of rocaglates or pateamines, eIF4A1:RNA
complexes are quite stable (> 50 mins) (Naineni et al., 2021). One question which arises is whether rocaglates and PatA
stabilize eIF4A on RNA sites normally occupied by eIF4A during its participation in the translation process and/or
whether they also induce unscheduled clamping of eIF4A1 to RNA sequences normally not visited by this helicase.
During the initiation process, eIF4A is present at the mRNA cap structure where one finds the eIF4F complex
bound to the cap (Sonenberg, 1981). Here, delivery of eIF4A to the RNA by eIF4F is thought to unwind local structure,
preparing the mRNA for ribosome recruitment. eIF4A1 molecules have also been captured (by UV crosslinking) 52
F I G U R E 5 Schematic illustration highlighting multiple ways by which RNA clamping of eIF4A by rocaglates has been shown to inhibit
translation. See text for details
bases downstream from the cap structure, suggesting that eIF4A may travel along the 50 leader region during initiation
(Lindqvist, Imataka, & Pelletier, 2008). eCLIP experiments performed on cells treated with the rocaglates, CR-1-31-B or
silvestrol, revealed a two-fold enrichment of reads aligned to the 50 leader region, and read density was enriched in
regions upstream of initiation codons (Chu et al., 2020). The results suggest that rocaglates cause clamping to sites
within the mRNA 50 leader region that are normally visited by eIF4A.
The mechanism of inhibition by rocaglates on translation is complex (Figure 5). First, rocaglate-induced clamped
complexes have been shown to block scanning ribosomes (Iwasaki et al., 2016). This results in (i) a reduction in the
translation of the main ORF, and (ii) if an eIF4A clamp is located 20–30 nucleotides downstream of an upstream initia-
tion codon, then translation of that uORF is increased. This is presumably due to the reduced scanning rate of the 40S
ribosome upon reaching the uORF start codon, allowing for increased sampling of codon:anticodon interactions at the
initiation codon. Rocaglates also induce clamping of eIF4F at the cap structure and this leads to a reduction in 43S PIC
recruitment (Chu et al., 2020). If ribosomes cannot load onto the mRNA template, this may be the primary route by
which translation initiation is inhibited. In the absence of 50 end polypurine sequences required for clamping of eIF4F,
then the scanning block may become important for inhibiting 43S complexes that have loaded onto the mRNA.
Rocaglate treatment of cells at high concentrations (200 nM) has also been shown to stabilize eIF4A onto ribosomes,
to the extent that eIF4A can be found trailing significantly into the heavy polysome portion of sucrose gradients follow-
ing sedimentation velocity centrifugation (Bordeleau et al., 2008). Given that rRNA consists of 95% of total cellular
RNA, it is not unreasonable to propose that this may act as a “sink” to sequester a significant pool of free eIF4A. At
some point, this could lead to depletion of eIF4A from the eIF4F complex and may explain a bystander effect that has
been reported where mRNAs recalcitrant to low concentrations of rocaglates become sensitized at higher levels (Chu
et al., 2020).
Recently, a 40S ribosome quality control pathway (iRQC) has been described in which a block in scanning leads to
43S PIC collisions within the 50 leader region. This activates ubiquitylation of uS3 and uS5 ribosomal proteins leading
to 40S subunit degradation (Garshott et al., 2021). Treatment of 293T cells with RocA or PatA for 2 h was found to
induce uS3 and uS5 ubiquitylation, presumably a consequence of ribosome collisions induced by eIF4A:RNA clamping
within the mRNA 50 leader region (Garshott et al., 2021). The impact of RocA and PatA on 40S ribosome stability under
such situations remains to be examined and iRQC activation may be an additional facet of how these compounds affect
initiation (Garshott et al., 2021).
eIF4A has recently been shown to limit the recruitment of RNAs to stress granules and reduce their formation
(Tauber et al., 2020). PatA perturbs these activities and reduces the dynamic relationship between P-body docking with
SGs and is thus expected to have a broader impact on RNA biology. Taken together, these results demonstrate that by
imparting gain-of-function properties to their targets, molecular staples can interdict the gene expression circuitry in
complex manners that are difficult to predict.
6 | C ON C L U S I ON
The current landscape of DEAD-box inhibitors is clearly sparse, with most helicases being drugless orphans. This repre-
sents an exciting opportunity, and we foresee much more effort dedicated to discovering small molecules that can be
used as chemical probes and possibly in drug development. We are strong proponents of demonstrating the selectivity
of target inhibition and would caution against the interpretation of biological results using compounds that have not
undergone rigorous validation. One powerful way of assigning selectivity is by undertaking genetic screens that identify
compound-resistant alleles. This genetic data, when coupled with biochemical data, provides a strong foundation for
supporting more complex biological experiments assessing compound activity in vivo.
As well, before the mechanism of action of rocaglates and pateamines was understood, the idea of screening for
compounds that could “staple” helicases to their nucleic acid substrate was not on the radar screen of many labs. Since
only one clamped eIF4F at the cap structure or (in principle) only one eIF4A molecule needs to be stapled within the 50
leader to induce a translational block, it becomes unnecessary to engage all eIF4A molecules to exert a biological effect
and this may explain the potency of these compounds. The interpretation of the biological activity of inhibitors that
induce “stapling” can be, however, more complex. Hence, knockdown or knockout experiments of the target may not
necessarily phenocopy compound activity and therefore cannot inform on compound target selectivity. Finally,
approaches that could engineer RNA targeting specificity into interfacial inhibitors would offer wonderful opportunities
to explore gene-specific regulation.
A U T H O R C ON T R I B U T I O NS
Sai kiran Naineni: Conceptualization (equal); visualization (equal); writing – original draft (supporting); writing –
review and editing (supporting). Francis Robert: Conceptualization (equal); visualization (equal); writing – original
draft (supporting); writing – review and editing (supporting). Bhushan Nagar: Validation (equal); writing – original
draft (supporting); writing – review and editing (supporting). Jerry Pelletier: Conceptualization (equal); funding
acquisition (lead); visualization (equal); writing – original draft (lead); writing – review and editing (lead).
FUNDING INFORMATION
We apologize to those authors whose work we did not cite due to space constraints. Work in the author's lab on this
topic is funded by a grant from the Canadian Institutes of Health Research (CIHR: FDN-148366).
CONFLICT OF INTEREST
The authors declare there is no conflict of interest.
DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data was generated or analyzed.
ORCID
Jerry Pelletier https://orcid.org/0000-0003-1963-6466
R EL ATE D WIR Es AR TI CL E
Modulating splicing with small molecular inhibitors of the spliceosome
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How to cite this article: Naineni, S. K., Robert, F., Nagar, B., & Pelletier, J. (2023). Targeting DEAD-box RNA
helicases: The emergence of molecular staples. WIREs RNA, 14(2), e1738. https://doi.org/10.1002/wrna.1738

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Received: 15 February 2022 Revised: 13 April 2022 Accepted: 16 April 2022

Targeting DEAD-box RNA helicases: The emergence of

Sai Kiran Naineni1 | Francis Robert1 | Bhushan Nagar1 | Jerry Pelletier1,2,3

This article is categorized under:

WIREs RNA. 2023;14:e1738. wires.wiley.com/rna © 2022 Wiley Periodicals LLC. 1 of 22

2 | REGULATION OF GENE EXPRESSION BY RNA H ELICASES

2.1 | Transcription, splicing, and nucleo-cytoplasmic transport

2.2 | Exon–junction complex formation

2.3 | Ribosome RNA processing

2.5 | Messenger RNA degradation

2.6 | MicroRNA-mediated silencing

2.7 | Cytoplasmic transport and storage

3.1 | Translation initiation

3.2 | Nonsense-mediated decay

3.3 | Ribosomal RNA biogenesis

4.1 | DDX2A/DDX2B (eIF4A1/eIF4A2)

5.1 | Molecular basis for eIF4A:RNA targeting

5.2 | Mechanism of translation inhibition

DATA AVAILABILITY STATEMENT

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