Biol. Chem.

2021; 402(5): 581–591

Review

Philipp Schult and Katrin Paeschke*

The DEAH helicase DHX36 and its role in

G-quadruplex-dependent processes
https://doi.org/10.1515/hsz-2020-0292 the structure (Tippana et al. 2014). A monovalent cation,
Received August 25, 2020; accepted September 24, 2020; preferably K+, is coordinated in the center between two
published online October 12, 2020 tetrads and further stabilizes the G4 (Figure 1A, C–F) (Sen
and Gilbert 1990; Williamson et al. 1989).
Abstract: DHX36 is a member of the DExD/H box heli-
G4 taxonomy is defined by the 5′-3′ directionality of the
case family, which comprises a large number of proteins
involved strands, which depends on the torsion angle of the
involved in various cellular functions. Recently, the
χ-glycosidic bond between ribose and guanine. In the syn-
function of DHX36 in the regulation of G-quadruplexes
conformation, the base is oriented towards the sugar and in
(G4s) was demonstrated. G4s are alternative nucleic acid
the anti-conformation it faces in the opposite direction
structures, which influence many cellular pathways on a
(Figure 1B). In parallel G4s, guanines are coordinated in an
transcriptional and post-transcriptional level. In this
anti-conformation and all strands are aligned in the same
review we provide an overview of the current knowledge
direction, while in antiparallel structures only the strands on
about DHX36 structure, substrate specificity, and
opposite sides are in the same orientation (Figures 1C, D).
mechanism of action based on the available models and
Lastly, the hybrid type constitutes a mixed assembly of par-
crystal structures. Moreover, we outline its multiple
allel and anti-parallel strands (Figure 1E) (Esposito et al.
functions in cellular homeostasis, immunity, and dis-
2007). Of note, most naturally occurring RNA G4s (rG4) are of
ease. Finally, we discuss the open questions and provide
the parallel type, because the charged 2′-hydroxyl interferes
potential directions for future research.
with the syn-orientation of the base (Fay et al. 2017), which is
Keywords: immunity; transcription; translation. not the case for DNA. Apart from unimolecular G4s, a variety
of intermolecular structures is also possible (Figure 1F). So far,
the relevance of G4 topology in vivo has not been determined.
Computational prediction algorithms for potential
Introduction: structure and function quadruplex-forming sequences (PQS) and G4-seq revealed
of G-quadruplexes more than 360,000 and 700,000 PQS in the human
genome, respectively (Chambers et al. 2015; Huppert and
G-quadruplexes (G4s) are secondary structures that can Balasubramanian 2005; Marsico et al. 2019). A subset (∼10
form in nucleic acid sequences with a high guanine con- 000) of these was validated by ChIPseq experiments
tent. In the presence of at least four runs of two consecutive (Hänsel-Hertsch et al. 2016). Moreover, rG4-seq analysis
guanines, separated by usually 1–12 other nucleotides, the identified ∼13,000 PQS in the human transcriptome (Kwok
guanines can associate via Hoogsteen base pairing (N1-H- et al. 2016). While individual structural confirmation of
O6 and C2-N-H-N7) to form stacks of planar tetrads many of these sites has not been achieved, their conser-
(Figure 1A) (Arnott et al. 1974; Gellert et al. 1962). The vation and location at functional regions indicate their
thermodynamic stability of this fold is positively influenced importance. PQS are overrepresented at promoters (Eddy
by the number of possible stacks and negatively by the et al. 2011; Huppert and Balasubramanian 2006), telomeres
length of the spacer sequences, which extend as loops from (Henderson et al. 1987) and 5′ and 3′ UTRs (Huppert et al.
2008; Maltby et al. 2019; Rouleau et al. 2017). Finally, PQS
were found in almost all species including pathogens, such
*Corresponding author: Katrin Paeschke, Department of Oncology, as bacteria and viruses (Saranathan and Vivekanandan
Hematology and Rheumatology, University Hospital Bonn, D-53127
2018), which further strengthens the assumption that G4s
Bonn, Germany, E-mail: [email protected].
https://orcid.org/0000-0003-3080-6745
function as an ancient regulatory element.
Philipp Schult, Department of Oncology, Hematology and In addition to their evolutionary conservation, genetic
Rheumatology, University Hospital Bonn, D-53127 Bonn, Germany and molecular analysis suggests defined cellular functions
582 P. Schult and K. Paeschke: DHX36 in G4 regulation

Figure 1: Graphical illustrations of G-quadruplex structures.

(A) Schematic representation of a planar tetrad formed by four guanines with a central monovalent cation (brown circle). (B) Anti and syn
conformation of guanosine. (C–F) Examples of possible G-quadruplex topologies. (G, H) NMR solution structures of the G4 in the 5′ region of
telomeric RNA and in the promoter region of myc. 3D models were created with ChimeraX. PDB entries are given in parentheses.

for a subset of G4s (Rhodes and Lipps 2015). Due to their family of enzymes that share a conserved helicase domain.
stability, and the finding that B-DNA is the preferred The C- and N-terminal regions adjacent to this common
conformation within cells, the current model is that G4 core are highly divergent and confer specificity for their
formation, function and unfolding is assisted by proteins. individual targets. DExD/H helicases perform roles in
Many DNA- and RNA-binding proteins were identified to almost all cellular processes. Apart from their canonical
associate with G4s (Brázda et al. 2014; Sauer and Paeschke activity of adenosine triphosphate (ATP)-dependent RNA
2017; Serikawa et al. 2018). Among these proteins, several and DNA unwinding they may act as RNA chaperones and
helicases have been identified that recognize and unwind in modulating ribonucleoprotein (RNP) complexes (Tanner
G4s in vitro and in vivo (Sauer and Paeschke 2017). and Linder 2001). Prominent members include DHX9 (also
Many of them belong to the group of DExD/H box RHA, NDHII), which has a wide range of functions from
helicases (Table 1) (Sauer and Paeschke 2017), a large transcription regulation, RNA processing and foreign
nucleic acid sensing (Aktaş et al. 2017; Fuller-Pace 2006;
Fullam and Schröder 2013; Kim et al. 2010) and DDX5 which
Table : Known G-specific DExD/H box helicases in human cells regulates miRNA processing, splicing as well as anti-viral
and their cellular function.
signaling (Cheng et al. 2018; Dardenne et al. 2014). Another
important cellular regulator of the G4 landscape is DHX36
Protein G-dependent Specificity Evidence
function (Creacy et al. 2008; Sauer et al. 2019; Vaughn et al. 2005).

BLM Regulation of DNA DNA Helicase assays (Huber

replication et al. )
DDX Expression of myc RNA ELISA, CD (Wu et al. ) DHX36 structure and enzymatic
DHX Regulation of RNA, DNA Helicase assays (Chakra-
transcription borty and Grosse ) functions
DDX IgG class switch RNA, DNA EMSA, pull down (Almeida
et al. )
DHX36 was first discovered as a protein binding to
DDX Regulation of RNA Pull down, helicase as-
translation says (McRae et al. )
AU-rich elements (AREs) and supporting RNA dead-
DHX Regulation of RNA, DNA PAR-CLIP (Sauer et al. enylation and degradation (Tran et al. 2004). Intriguingly,
translation/ ) it was demonstrated that it binds to G4s with extraordi-
transcription narily high affinity for RNA G4s(∼39 pM) and DNA G4s
FANCJ Regulation of DNA EMSA, helicase assay (Wu (∼77 pM) (Creacy et al. 2008). DHX36 unwinds G4 struc-
transcription and Spies )
tures with a much higher efficiency than double-stranded
 P. Schult and K. Paeschke: DHX36 in G4 regulation 583

(ds) DNA (Vaughn et al. 2005; Yangyuoru et al. 2017). The function and subcellular localization (Chalupníková et al.
catalytic activity of DHX36 is dependent on the thermo- 2008; M.C. Chen et al. 2018; W.-F. Chen et al. 2018;
stability of the G4. More stable G4s are unwound much Srinivasan et al. 2020).
slower by DHX36, which may fine-tune the regulatory Like all members of the DExD/H family, the core
functions of G4s in vivo (Chen et al. 2015). DHX36 has a helicase portion is comprised of two RecA-like folds
specific N-terminal region which harbors a Gly-rich region (RecA1 and 2; Figure 2A, B). These are defined by their
and a conserved nucleic acid-binding motif (DHX36-spe- ATPase structure of a central beta sheet surrounded by
cific motif [DSM]; Figure 2A, B) (Chalupníková et al. 2008; alpha helices. Within the beta strands are the conserved
M.C. Chen et al. 2018; W.-F. Chen et al. 2018). The latter Walker A (GxxxxGKT/S; XXXX: variable quartett) and
was identified as the region conferring the G4-binding Walker B (hhhhD; h: hydrophobic amino acid) motifs,
properties of DHX36 (Lattmann et al. 2010; Meier et al. which coordinate the γ-phosphate of an ATP and a Mg2+
2013). In this study it was also shown that the N-terminal ion, respectively. The hydrolysis of ATP leads to a sig-
domain has two helices (α1 and α2) followed by an un- nificant rearrangement of the helicase, from a closed to an
structured linker region (Figure 2B). The N-terminal helix open state, in which RecA2 is rotated away from RecA1.
α1 is positioned on top of the terminal G-plane of a G4 This movement relocates RecA2 relative to the template by
structure creating a hydrophobic interface exposing one nucleotide and flips the 5th nucleotide in the channel
nonpolar amino acids (Figure 2C). The exposed hydro- so that it stacks against a conserved β-hairpin (M.C. Chen
phobic surface of the G-tetrad is thermodynamically un- et al. 2018; W.-F. Chen et al. 2018).
favorable in solution. This is mitigated by enzyme binding The winged-like (WL), ratchet-like (RL), and
and provides a possible explanation for the high target oligosaccharide-binding-fold-like (OL) subdomains
affinity of DHX36. Moreover, DHX36 exhibits a strong (Figure 2A, B) make additional contacts to the single-
preference towards binding parallel G4s (Smaldino et al. stranded (ss) nucleic acid within the binding channel, but
2015; Yangyuoru et al. 2017). One reason might be that are not significantly rearranged during this movement
antiparallel or hybrid types expose polar surfaces via the (M.C. Chen et al. 2018; W.-F. Chen et al. 2018).
loops masking the terminal G-planes, which reduces the Solving the structure of DHX36 from different species
entropic benefit and may sterically hinder DSM binding. with and without bound G4, highlighted the role of exposed
Although important for G4 interaction, the N-terminal polar amino acids of the RecA2 subdomain in interacting
domain is not sufficient to disrupt the G4, which is only with the phosphate backbone of the G4 (M.C. Chen et al.
possible in combination with the conserved DExD/H 2018; W.-F. Chen et al. 2018). A distal loop of the OL region
helicase domain (Figure 2A, B). The N-terminus confers (OI) was also suggested to be involved in G4 positioning
further target specificity and is essential for enzymatic by interacting with the first single-stranded nucleotide

Figure 2: Graphical illustration of DHX36.

(A) Schematic of the domain organization
of human DHX36. (B) Crystal structure of
human DHX36 in complex with myc G4 DNA.
(C) Detailed view on the hydrophobic
interface between G4 and DSM. (D) View
inside the substrate tunnel with bound
ssDNA. (E) Simplified model of DHX36
enzyme activity exemplified by movements
of the helicase core. Note that structures of
the related helicase Prp43 were chosen as
surrogate for the ATP and adenosine
diphosphate (ADP) bound states, as these
are not yet available for DHX36. PDB entries
are given in parentheses.
584 P. Schult and K. Paeschke: DHX36 in G4 regulation

preceding the G4 (M.C. Chen et al. 2018; W.-F. Chen et al. a time (Figure 2E) (W.-F. Chen et al. 2018). This repetitive
2018). The nucleic acid: DHX36 interface in the single- motion can be observed in single-molecule FRET experi-
stranded section adjacent to the G4 is mainly formed by ments (Tippana et al. 2019).
hydrogen bonding of the DSM and the OI with the phosphate DHX36 also features a C-terminal extension (CTE,
backbone (M.C. Chen et al. 2018; W.-F. Chen et al. 2018). This Figure 2A). It has been observed that rotation of the CTE is
may explain the substrate promiscuity of DHX36 for binding necessary for target binding and enzymatic activity of
RNA, DNA, or even chimeric targets (Tippana et al. 2019). several DEAH helicases, in contrast to other families (Chen
Together, these parts of DHX36 form a pocket with charged and Ferré-D’Amaré 2017). The CTE is conserved among
and hydrophobic surfaces enclosing the G4 structure. orthologues of DHX36 in different species to a higher de-
The template channel of DHX36 can only accommo- gree than the N-terminus (Lattmann et al. 2010), but its
date single-stranded nucleic acids (Figure 2D) (M.C. Chen function has not been studied, yet. This domain and its
et al. 2018; W.-F. Chen et al. 2018), but the binding affinity biological relevance are of particular interest, because one
to unstructured targets is low and sequence-dependent unsolved question in the G4 helicase field is why almost all
(Creacy et al. 2008; Giri et al. 2011; Yangyuoru et al. 2017). helicases can unwind G4 structures and how these heli-
G4s are recognized with similar affinity, whether in cases gain their specificity. Of note, the closest mammalian
conjunction with a single-stranded region or by itself homolog of the CTE is found in YTHDC2, another DExD/H
(M.C. Chen et al. 2018; W.-F. Chen et al. 2018). In contrast, helicase (Bailey et al. 2017; Hazra et al. 2019), which rec-
the helicase activity of DHX36 requires a 3′ tail of 9 nt ognizes methylated adenosines (m6A). These data,
(Tippana et al. 2016). 5′-tailed G4s are not unfolded effi- together with the finding that G4s are proposed to be target
ciently by DHX36 indicating its 3′–5′ helicase activity structures that facilitate m6A modifications (Fleming et al.
(Gueddouda et al. 2017). This is corroborated by the fact 2019), leads to the speculation that DHX36 might also
that positioning the ss DNA/RNA within the enzyme is recognize specific G4s that are m6A modified.
crucial for the domain rearrangement upon ATP hydrolysis
(Gueddouda et al. 2017; Tippana et al. 2019; Yangyuoru
et al. 2017). However, the structures for the ATP + nucleic
acid to adenosine diphosphate (ADP) state transition have
DHX36 is a master regulator of
not been determined, yet, but were inferred from structures cellular homeostasis
of related enzymes.
In conclusion, the current model for G4 disruption G4s modulate various cellular functions (Bochman et al.
is as follows: initial binding is achieved by contacts at 2012; Spiegel et al. 2019). Accordingly, molecular and ge-
the G4 structure and single-stranded downstream se- netic experiments have been performed to understand the
quences, which leads to considerable translocations in biological function and relevance of DHX36 binding to G4s.
the RecA-like and OB subdomains. The motion from These analyses point towards a model that DHX36 is a
the ground to nucleic-acid-induced transition-state posi- multifunctional helicase that acts on DNA and RNA G4s
tions the 5th nucleotide at the RecA2 hairpin and the and supports various biological pathways. DHX36 is
OI-loop at the first nucleotide downstream of the G4 (M. C. expressed in two isoforms and can be localized in the nu-
Chen et al. 2018; W.-F. Chen et al. 2018; Srinivasan et al. cleus and cytoplasm (Iwamoto et al. 2008; Sauer et al.
2020; Tippana et al. 2016). This was reported to destabilize 2019). The predominant localization seems to depend on
the G4 structure by exerting a pulling force on the sugar- the cell type and the expressed isoform (Iwamoto et al.
phosphate backbone of the stack in an ATP-independent 2008; Sauer et al. 2019; Tran et al. 2004). Functionally, the
fashion (M.C. Chen et al. 2018; W.-F. Chen et al. 2018). isoforms have also been shown to affect certain target
However, this interpretation was challenged, because mRNAs differently (Booy et al. 2016; Tran et al. 2004).
the utilized folding conditions might have caused the DHX36 has been associated with transcriptional acti-
observed distortion of the G4 (Guo et al. 2019). Based on vation of several genes involved in cell growth and differ-
model predictions and studies of related enzymes it was entiation by binding and resolving G4s in their respective
suggested that ATP-binding results in the transition to a promoters (Chashchina et al. 2019; Gao et al. 2015; Giri et al.
closed state of RecA1 and A2 reorienting the RecA2 hairpin 2011; Huang et al. 2011; Iwamoto et al. 2008; Kim et al. 2011;
back to its original position (M.C. Chen et al. 2018; W.-F. Schlag et al. 2020). This is of importance, because actively
Chen et al. 2018; He et al. 2010). ATP hydrolysis enables a transcribed DNA is temporarily in a single-stranded state
RecA domain shift that pulls the template in a 3′–5′ di- and therefore prone to form secondary structures. Once
rection through the nucleic acid tunnel, one nucleotide at formed within promoters, G4 can either decrease
 P. Schult and K. Paeschke: DHX36 in G4 regulation 585

transcription by blocking DNA polymerase II (PolII) (Eddy other helicases can unfold these G4s and by this modulate
et al. 2011), or support transcription by recruiting proteins telomerase function (Smaldino et al. 2015).
that effect transcription itself (Cogoi et al. 2010; Raiber et al. A large body of evidence has been gathered for the
2011). Apart from the interrupted transcription, an internal functions of DHX36 in post-transcriptional regulation,
arrest of PolII has been associated with genomic instability such as translation and RNA degradation (Murat et al. 2018;
via a topoisomerase 2-dependent mechanism (Magis et al. Nie et al. 2015; Tran et al. 2004). In a global approach it has
2018; Pipier et al. 2020). This suggests a critical role of been shown that G4-containing mRNAs are located to
DHX36 in maintaining genomic integrity under physio- cytoplasmic stress granules (SGs) upon external stress
logical and stress conditions. One of the major guardians of signals (e.g., arsenite) (Sauer et al. 2019). DHX36 is
genome stability is TP53 (or p53). TP53 blocks mitotic ac- recruited to SGs and releases target mRNAs, which then
tivity as soon as genomic instability is detected and thereby become translational competent (Vester et al. 2018).
prevents excessive mutation of the genome upon replica- Moreover, DHX36 knock-out cells are prone to form SGs,
tion (Williams and Schumacher 2016). It was demonstrated even without external stressors (Sauer et al. 2019). There-
that DHX36 controls the TP53-dependent DNA damage fore, it can be assumed that DHX36 is necessary to rees-
response, which is activated upon ultraviolet (UV) light- tablish cellular homeostasis after stress. To this end, it is
induced DNA cross-linking or disruption (Newman et al. also noteworthy that only a subset of cytoplasmic RNAs are
2017). DHX36 interacts with a G4 in the 3′-UTR poly- actively targeted to SG upon stress conditions (Sauer et al.
adenylation site of the TP53 pre-mRNA and stimulates 2019).
3′-end processing, which leads to selective ongoing protein Most of these RNAs have been found to contain AREs,
production (Newman et al. 2017). which may control their recruitment by an interaction
Another line of evidence connecting DHX36 function with the proteins TIA/TIAR, as well as GC-rich stretches
to genome stability is based on its involvement in telo- prone to form G4s (Namkoong et al. 2018). This may
mere maintenance. Telomeres are the terminal structures indeed provide another potential clue for DHX36 target
of most eukaryotic chromosomes that protect chromo- recognition within SGs, considering that AREs were the
somes from degradation, end-to-end joining or recogni- first specific sequence motifs associated with the helicase.
tion as double-strand breaks (DSBs) among other This is corroborated by a stronger activation of ATPase
functions (Blackburn et al. 2015; Lange 2009). Telomeres activity upon binding to single-stranded U-rich oligonu-
are maintained by telomerase, a reverse transcriptase cleotides in vitro, compared to other sequences (Tran et al.
harboring an internal RNA template for the synthesis of 2004).
telomeric repeats. In the 5′ portion of this RNA, a stem- Despite higher translation levels, DHX36 target mRNAs
loop structure (P1) is providing the template boundary for also exhibited decreased stability (Sauer et al. 2019). It has
accurate termination of transcription (Chen et al. 2000; not been clarified, whether this is merely due to the release
Webb and Zakian 2015). Due to its high guanine-content, a from SGs, where stored RNAs are protected from decay by
G4 can also form within this region, which was shown to association with protein complexes (Decker and Parker
disrupt the P1 helix (Gros et al. 2008; Li et al. 2007). Due to 2012), or whether DHX36 plays an active role in fate
the central role of P1, modifications such as structure determination of these mRNAs.
formation within this region are predicted to alter telo- Considering the enrichment of ARE-containing mRNAs
merase function and accuracy. These findings lead to the in SGs, a mechanism that may explain the dichotomy of
model that G4 formation within the P1 helix required increased translation versus reduced stability (Sauer et al.
unwinding to ensure the proper template boundary. 2019) is exemplified by the regulation of the Nkx2-5 mRNA.
Indeed, G4 formation and DHX36 are both connected to Here, the resolution of a G4 in the 5′ UTR leads to higher
telomere length maintenance, because disruption of the translation, while binding of DHX36 to an ARE in the 3′ UTR
G4 motif as well as down regulation of DHX36 by siRNA targets the mRNA for degradation (Nie et al. 2015). This
lead to reduced telomerase function and consequently supports other studies, which found DHX36-binding
shortened telomeres (Sexton and Collins 2011). DHX36 has associated with 3′–5′ exosome activation after binding to
been shown to interact with G4s within the telomerase AREs in the urokinase-type plasminogen activator (uPA)
RNA (Booy et al. 2012; Lattmann et al. 2011; Sexton and mRNA (Tran et al. 2004). DHX36 also interacts with the
Collins 2011). Moreover, the current paradigm also sug- microRNA (miR) machinery on several levels to destabilize
gests G4s as a protective cap to regulate telomerase target mRNAs (Bicker et al. 2013; Booy et al. 2013). Inter-
binding to telomeres (Jurikova et al. 2020). DHX36 and estingly, under steady-state conditions, target transcript
586 P. Schult and K. Paeschke: DHX36 in G4 regulation

stability seems to be less affected by a DHX36 knock-down, (Tsai and Lloyd 2014). In combination with the involve-
suggesting different regulatory mechanisms involving ment of DHX36 in SG regulation discussed above, these
specific factors (Iwamoto et al. 2008). findings raise the questions, if G4 formation during viral
infection affects DHX36 expression and function.
Differential regulation of DHX36 among other
Disease potential of DHX36 conserved DExD/H helicases was shown in zebrafish and
carp after viral infection (Mojzesz et al. 2020). This is in
mutation, dysregulation or loss agreement with the fact that viral RNA (vRNA) and its
surrogate poly(I:C) are effectively sensed by a number of
DHX36 is involved in the regulation of many crucial path-
DExD/H helicases (including DHX36), which facilitate
ways for cellular maintenance. TP53 is a key protein for the
maximal efficiency of the subsequent immune response
control of genome replication upon DNA damage and
(Fullam and Schröder 2013). One of these complexes con-
stress conditions and is regulated by DHX36 in a post-
sists of RIG-I and DHX36 (Yoo et al. 2014). vRNA binding
transcriptional manner (Newman et al. 2017), as discussed
triggers autophosphorylation and activation of the double-
above. Hence, loss of DHX36 may decrease TP53 levels in
stranded RNA-dependent protein kinase PKR. The latter
response to genome instability leading to an increased
depends on the ATPase activity of DHX36, which indicates
mutation rate during DNA replication.
helicase activity (Yoo et al. 2014). Direct DHX36 binding to
In addition to MYC (a family of regulatory genes and
several segments of the Influenza virus genome has been
proto-oncogenes that encode for transcription factors) (Giri
reported to influence RIG-I-dependent avSG formation
et al. 2011), the tyrosine kinase KIT complements the panel of
(Yoo et al. 2014). However, a direct involvement of G4s has
proto-oncogenes that contain G4 motifs in their promoters
not been examined, yet.
(Rankin et al. 2005). These genes are critically involved in
In murine dendritic cells (mDCs) DHX36 is found in a
cell growth and therefore need to be tightly regulated. There
multi-helicase complex with DDX1 and DDX21, which acts
is evidence that MYC promoter activity is modulated by a G4.
as a cytoplasmic sensor of viral double-stranded RNA
However, while DHX36 can unwind this structure in vitro, it
(dsRNA) (Zhang et al. 2011). DDX1 seems to be mainly
is currently not clear if it is directly involved in MYC
responsible for dsRNA binding, whereas DHX36 and DDX21
expression in vivo. In contrast, for KIT a DHX36-dependent
do not directly interact with poly(I:C) but are necessary for
upregulation of transcription has been reported (Gao et al.
the antiviral response (Zhang et al. 2011). Importantly, all
2015).
these helicases are known for their capability to bind and
Previously, it was discovered that DHX36 is recruited
unwind G4s (Almeida et al. 2018; Creacy et al. 2008; McRae
and inhibited by G4 structures of the long non-coding RNA
et al. 2017). However, also in this case a clear link to viral
(lncRNA) GSEC (G-quadruplex-forming sequence contain-
G4s remains to be established.
ing lncRNA), which leads to high motility of colon cancer
Lastly, DHX36 together with DHX9 is involved in the
cells (Matsumura et al. 2016). Moreover, GSEC was found to
detection of pathogenic DNA in the cytoplasm of human DCs.
be upregulated in various other cancer types, which may
This was demonstrated by transfections with synthetic
suggest a common DHX36-dependent mechanism in
mimics of microbial CpG islands (CpG-A and B) and with a
carcinogenesis (Matsumura et al. 2016). Accordingly,
DNA virus (Herpes Simplex Virus-1, HSV-1) (Kim et al. 2010).
DHX36 has been classified as an oncogene (Davoli et al.
CpG-A is detected by DHX36, while DHX9 recognizes CpG-B
2013) and is upregulated in multiple human cancers (Thul
and Lindskog 2017) and cancer cell lines (Nusinow et al. (Kim et al. 2010). CpG-A oligonucleotides are characterized
2020). This may hint towards a so far unexplored roles of by flanking oligo-G stretches (5′-GGGGGACGATCGTCGGG-
DHX36 in these pathologies. GGG-3′) in contrast to CpG-B (5′-TCGTCGTTTTGTCGTTTTGT-
CGTT-3′). Accordingly, CpG-A oligonucleotides form highly
stable intermolecular quadruplexes (Kerkmann et al. 2005),
DHX36 and other helicases may link G4s and which could link G4s to DHX36-specific innate immune re-
antiviral immune responses sponses. Concomitantly, the DHX36-dependent activity
against HSV-1 (Kim et al. 2010) and the high amount of PQS in
G4s are overrepresented in most viral genomes (Lavezzo the HSV-1 genome (Biswas et al. 2016) may point in the same
et al. 2018), but their impact on antiviral immunity remains direction. Additional research is needed to find further clues
elusive. Formation of antiviral stress granules (avSG) has if and how G4s of pathogens trigger a DHX36-dependent
been shown to be a common consequence of virus infection immune activation.
 P. Schult and K. Paeschke: DHX36 in G4 regulation 587

Potential direct DHX36 interactions (M.C. Chen et al. 2018; W.-F. Chen et al. 2018; Lattmann
et al. 2010). However, it affects the intracellular localiza-
with viruses tion to SG in human cells (Chalupníková et al. 2008),
which may suggest species-dependent protein:protein in-
Apart from governing antiviral innate immune responses, teractions. Similarly, the function of the CTE is entirely
DHX36 may also have a direct influence on the viral life unstudied. The high sequence similarity among species
cycle, as many related DExH/D helicases do (Biegel et al. points towards a conserved biological role (Lattmann et al.
2017; Cheng et al. 2018; Grünvogel et al. 2015; Matkovic 2010).
et al. 2018). However, there is only sparse information Furthermore, the question of the molecular de-
available in current literature about such interactions and terminants for target specificity is not finally answered. The
any functional information is lacking (Calderone et al. structure and charge-based binding model for G4s can
2014). explain the preference of DHX36 for certain types of G4s,
In addition to the interaction with some of the Influenza but additional sequence specificity for the downstream
A virus genome segments, DHX36 was also shown to bind single stranded region seems likely. As U-rich sequences
directly to the Influenza nucleoprotein (NP), non-structural show enhanced binding and ATPase activation (Tran et al.
protein 1 (NS1) and the polymerase subdomain (PA) by co- 2004), it may be interesting to assess whether consensus
immunoprecipitation (Wang et al. 2017). Another connec- patterns can guide DHX36 to specific targets. This will
tion was found with the nucleocapsid protein of the porcine also contribute towards the understanding of the regula-
reproductive and respiratory syndrome virus (PRRSV) tion of other G4-binding proteins. Along this line it will be
(Jourdan et al. 2012). DHX36 also binds to the Rev response interesting to address how the associated 3′single-stranded
element (RRE) of HIV, a highly structured RNA region that regions and loops, as well as the topology of bound G4s
controls nuclear export of unspliced viral mRNAs (Naji et al. (Tippana et al. 2014) may change their recognition by
2011). Although the RRE contains several G-rich sequences, different complexes and thereby determine specific
no G4 has been validated, yet. HIV relies on its nucleocapsid downstream effects.
protein to destabilize G4s in its genome for efficient reverse Apart from these fundamental questions, future
transcription (Butovskaya et al. 2019). The viral reverse research should also investigate the influence of DHX36 on
transcriptase is stalled by G4s, which may lead to a template regulatory networks of oncogenesis. Foremost, global an-
switch and recombination events associated with immune alyses will be required investigating transcription and
evasion (Shen et al. 2009). Finally, several conserved G4s translation levels of known oncogenes in DHX36-depleted
were detected in the promoters of lentiviruses, which control cells. In this context it would be interesting to clarify how
gene transcription in a similar fashion as cellular genes naturally occurring mutations of DHX36 are involved in
(Perrone et al. 2013, 2017). Due to these findings it is cell transformation. Moreover, large gaps remain in the
tempting to speculate that DHX36 may also modulate these knowledge about direct interactions of DHX36 with viral
processes. nucleic acids and its role in viral infection. Considering the
In conclusion, a vast amount of knowledge is still to be involvement of DHX36 in several immune sensor com-
gained by investigating the host:pathogen interface of plexes (Fullam and Schröder 2013; Yoo et al. 2014; Zhang
DHX36. et al. 2011), makes DHX36 a prime candidate for further
investigations.
Finally, the discovery of a link between G4 structure
and chemical modifications has opened a whole new field
Perspectives and open questions of research. It will be interesting to learn how these path-
ways are functionally linked and if DHX36 may shape the
DHX36 is a well-characterized protein, but due to its landscape of the epitranscriptome.
multitude of functions and the fact that G4 biology has only In conclusion, DHX36 is an important regulator of
recently gained its current level of interest, the cellular many cellular pathways and several open questions
characterization of DHX36 is far from complete. It is still remain to be answered, which will contribute greatly to our
unclear, which molecular role the N-terminal glycine-rich understanding of tissue homeostasis, stress regulation,
region or the C-terminal extension play in the enzymatic and immune responses.
activity. Notably, the glycine-rich stretch is completely
absent in Drosophila (Lattmann et al. 2010) and binding of Acknowledgments: We thank Stefan Juranek for careful
the human enzyme to G4s is not dependent on this segment reading of the manuscript. Research in the Paeschke
588 P. Schult and K. Paeschke: DHX36 in G4 regulation

laboratory is funded by an ERC Stg Grant (638988-G4DSB) promotes the formation of the P1 helix template boundary.
and by the Deutsche Forschungsgemeinschaft (DFG, Nucleic Acids Res. 40: 4110–4124.
Booy, E.P., Howard, R., Marushchak, O., Ariyo, E.O., Meier, M.,
German Research Foundation): Project-ID 369799452 –
Novakowski, S.K., Deo, S.R., Dzananovic, E., Stetefeld, J., and
TRR237” and under Germany’s Excellence Strategy – McKenna, S.A. (2013). The RNA helicase RHAU (DHX36)
EXC2151 – 390873048. suppresses expression of the transcription factor PITX1. Nucleic
Author contribution: All the authors have accepted Acids Res. 42: 3346–3361.
responsibility for the entire content of this submitted Booy, E.P., McRae, E.K.S., Howard, R., Deo, S.R., Ariyo, E.O.,
Dzananovic, E., Meier, M., Stetefeld, J., and McKenna, S.A.
manuscript and approved submission.
(2016). RNA helicase associated with AU-rich element (RHAU/
Research funding: Research in the Paeschke laboratory is
DHX36) interacts with the 3′-tail of the long non-coding RNA
funded by an ERC Stg Grant (638988-G4DSB) and by the BC200 (BCYRN1). J. Biol. Chem. 291: 5355–5372.
Deutsche Forschungsgemeinschaft (DFG, German Brázda, V., Hároníková, L., Liao, J.C.C., and Fojta, M. (2014). DNA and
Research Foundation): Project-ID 369799452 – TRR237” RNA quadruplex-binding proteins. Int. J. Mol. Sci. 15:
and under Germany’s Excellence Strategy – EXC2151 – 17493–17517.
Butovskaya, E., Soldà, P., Scalabrin, M., Nadai, M., and Richter, S.N.
390873048.
(2019). HIV-1 nucleocapsid protein unfolds stable RNA
Conflict of interest statement: The authors declare no G-quadruplexes in the viral genome and is inhibited by
conflicts of interest regarding this article. G-quadruplex ligands. ACS Infect. Dis. 5: 2127–2135.
Calderone, A., Licata, L., and Cesareni, G. (2014). VirusMentha: a new
resource for virus-host protein interactions. Nucleic Acids Res.
43: D588–D592.
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