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11.2 Regulation and Dysregulation of RNA–Protein Interactions 295
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transcripts harboring a (CUG)n(n = fty to a few thousand) expanded repeat in
the 3′UTR [118–122]. In normal cells, DMPK mRNA is transcribed, transported to
nuclear speckles, and then exported from the nucleus. The (CUG)
repeats prevent
exp
its entry into nuclear speckles, and instead causes the RNA to accumulate in separate and distinct nuclear foci, and in the process, sequester the ZnF-containing RBP
Muscleblind-like protein 1 (Mbnl1) to those foci. Subsequently, Mbnl1 is prohibited
from carrying out its role as an essential regulator of alternative splicing, resulting
in many mis-spliced RNAs in DM1. The (CUG)
nuclear foci are suggested to be
exp
the source of pathology in DM1 with an increased number of repeats correlative
with disease severity due to enhanced ability to accumulate and recruit more Mbnl1
proteins [123]. This is also a case to exemplify a disease in which the driver of the
disease is a toxic gain of function in the RNA, which causes an RBPs’ functions to
lead to symptoms rather than the RBP being the main pathological driver.
11.2.4 The RBP is Lost and Wreaks Havoc on the Cell
As mentioned in Section 11.1, localization of an RBP plays a large role in dictating which RNAs it may interact with where mis-localization of the RBP will
cause adverse eects through binding to o-target RNAs. Various mutations in
RRM-containing protein TDP-43 promote mislocalization to subcellular locations
in the cytoplasm, while others can cause retention in the nucleus [110, 124, 125].
Previous reports have shown that mutant forms of TDP-43 increased mislocalization
to the mitochondria, thereby leading to the repression of the expression of mitochondrial RNAs [126]. Similarly, the KH domain-containing RBP Src associated
in mitosis of 68 kDa (Sam68) undergoes various posttranslational modications,
which greatly inuence its biochemical properties, ne tuning its subcellular
localization, and ability to interact with signaling proteins and target RNAs. Sam68,
an almost exclusively nuclear protein containing a nuclear localization signal, is
localized to the cytoplasm in some cells and plays a role in translational regulation
of target mRNAs [127, 128]. However, various cancer types show that higher
Sam68 expression in the cytoplasm contributes to tumor progression and metastasis
[129]. Upregulation and increased cytoplasmic localization of Sam68 signicantly
correlate with pathological grade in patients with renal cell carcinoma [130],
tumor-nodule metastasis and other outcomes in breast cancer patients [131], and
lymph node metastasis in patients with early-stage cervical cancer [131].
11.2.5 RBPs Dictate Which mRNAs are Translated, Favoring their Tox i c
Friends
One of the most direct impacts RBPs have on regulating gene expression is through
interacting with transcripts in the cytoplasm, where they inuence stability and
translational eciency of target RNAs.The dsRBD-containing RBP Staufen1 (Stau1)
is a multifunctional protein that regulates all aspects of RNA metabolism; here, we
will discuss its role in translation and decay and its contribution to cancer [132].
Stau1 binds to the 5′UTR of target RNAs, simultaneously with the ribosome, and
296 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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transports an mRNA to the site of translation in the rough endoplasmic reticulum
to promote protein synthesis [133]. Stau1 can also bind to regulatory motifs in the
3′UTR of transcripts to promote mRNA stabilization, enhancing translation [134].
In undierentiated C2C12 myoblasts, for example, Stau1 directly binds to and stabilizes the 3′UTR of Dvl2 mRNA, promoting cell proliferation. During myogenesis, Stau1 levels become reduced, and along with it the half-life of Dvl2 mRNA,
prompting the induction of myogenic dierentiation [135]. Stau1 can also bind to
double-stranded regions downstream of the stop codon in the 3′UTR of transcripts,
and through direct interaction with the ATP-dependent helicase UPF1 to enhance
its activity, promote Stau1-mediated decay [136]. Through these various functions,
Stau1 can support cellular proliferation, tumor growth, and metastasis in cancer
through stabilization of transcripts promoting those functions such as SIRT1 mRNA
[137]. Stau1-mediated decay may also contribute to increased growth of cancer cells
through targeting proteins, which act as transcription and tumor suppressors such
as ZNF331 [138]. Alternatively, Stau1 has the capacity to be able to inhibit cancer
growth by promoting the decay of transcription factors such as RAX2 [139]. Like
the previous examples highlighted, the role of Stau1 in disease progression is context dependent, stressing the importance of maintaining proper regulation of the
broader network of interactions between RNA and RBPs.
11.2.6 RBPs and RNA Become Very Clique-y, Form Their Own Complex
and Cause Stress to the Rest of the Cell
The RBP fused in sarcoma (FUS), containing both an RRM and ZnF, is highly
mutated in ALS patients, where mutations largely occur in prion-like domains of
the protein priming it for aggregation [140]. Furthermore, previous studies have
found that while nuclear FUS does not incorporate into stress granules, mutant
FUS can bind and sequester nuclear localized FUS to cytoplasmic stress granules
[141]. More broadly, many RBPs display a propensity to assemble in membrane-less
compartments through the process of liquid–liquid phase separation (LLPS) [142].
These compartments, composed primarily of RBPs and RNAs, are dynamic and
reversible and allow for the exchange of macromolecules with their surrounding
environment [143]. LLPS particles are often held together by the accumulation of
multiple weak interactions between RNAs and RBPs [143–145]. These interactions
generally engage the low complexity intrinsically disordered regions that are
characteristic of many RBPs. Various RNP complexes, such as the nucleolus,
stress granules, P-bodies, paraspeckles, Cajal bodies, and others, are proposed
to form following the principles of phase separation [146–149]. In the case of
some disease states, LLPS condensates can transition to more viscous states with
reduced uidity and even further to more dense pathological aggregates [150, 151].
This is the case for many degenerative brain diseases which are characterized by
plaque-like aggregates composed of RBPs. Additionally, many disease-associated
RBPs contain disordered prion-like domains, facilitating PPIs and accelerating
liquid to solid-phase transitions.
11.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate 297
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11.3 Experimental Methods to Detect and Screen
for Small Molecules that Modulate RNA–Protein
Interactions
Due to the increasing evidence of RPI dysregulation in diseases, targeting RBPs and
RPIs has surfaced as a new subject in RNA-targeted drug discovery [152]. The last
two decades have uncovered many networks of RPIs and resulted in the application
of existing methods and development of new technologies for detecting and validating RPIs in vitro and in cellulo. Here, we will highlight methods that can be used
to detect and validate RPIs, as well as discover small-molecule modulators of RPIs.
Examples of hits identied using these approaches will also be discussed and can be
found in Table 11.2.
11.3.1 In vitro Fluorescence-Based Assays
Fluorescence-based assays, including uorescence polarization (FP)- and uorescence resonance energy transfer (FRET)-based methods (Figure 11.10), provide
quantitative measures of an RPI resulting in an ability to measure accurate binding
anities and determine IC50values for inhibitors. SPR and ITC are also powerful
tools for the quantitative characterization of RPIs [153–157]; however, these techniques require specialized instrumentation and are not considered high-throughput
for inhibitor discovery. FP utilizes a uorophore-labeled biomolecule, in this case
a uorescently labeled RNA probe, which tumbles in solution at a rate dependent
upon the size of the molecule [157–162]. An RPI is detectable when there are
changes in polarization caused by protein binding to the uorescently labeled
RNA, slowing the rotation and emitting an FP signal. FRET, on the other hand,
utilizes donor and acceptor uorophore pairs where excitation of the donor uorophore results in the transfer of energy to the acceptor uorophore when in close
proximity [163–165]. In the case of an RPI, the donor and acceptor uorophores
are conjugated to the RNA and protein. Both FP and FRET are compatible with
high-throughput small-molecule screening and accordingly have been used to
identify inhibitors of RPIs. Select examples of RBP-binding compounds discovered
using these approaches include multiple inhibitors of Lin28 [164, 166] binding to
pre-let-7 substrates and disrupters of the binding of Msi1/2 [167] and eIF4E [168] to
RNAs comprising consensus binding sequences (Table 11.2).
11.3.2 In vitro Chemiluminescence-Based Assays
Although robust and high-throughput, uorescence-based assays are prone
to compound interference by autouorescent molecules or compounds that
can function as uorescence quenchers [169]. To overcome these limitations,
chemiluminescence-based detection assays for RPIs have been developed, including catalytic enzyme-linked click chemistry assay (cat-ELCCA) and split enzyme
assay (SEA) technology (Figure 11.11) [170, 171]. Both of these methods are plate
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Table 11.2 Select examples of RBP-binding compounds discovered using assays
mentioned in Sections 11.3.1–11.3.2.
Compound Structure Target Activity Assay
PH-43
Ro 08-2750
eFFECTOR 634
compound 1
OHC
MeN
S
N
O
HO
N
O
O
OH
CO2H
Lin28 IC50:5μM
Kd:16μM
Msi2 IC50:2.7μM
N
N
N
CN
N
F
C
3
NH
O
O
N
N
HO2C
O
Me
Kd:11μM
Cl
O
S
eIF4E 5.6 μM FP
Me
N
CO2H
Lin28 IC50:4μM
FP
FP
FRET
Kd:3.5μM
N
O2N
N
CCG-233094
based, amenable for high-throughput screening, and rely on click chemistry-based
detection strategies.
Cat-ELCCA has been applied to multiple biochemical systems, including monitoring the enzymatic activities of ghrelin O-acyltransferase (GOAT) [172] and Dicer
[173–175], and biomolecular interactions including RPIs (e.g. Lin28-pre-let-7 interaction [176]) and PPIs (e.g. eIF4E-4E-BP1 PPI [177]). For RPIs, a biotinylated-RBP
Me
O
O
O O
S
N
N
H
HN
N O
F
S
O O
Me
Lin28 IC50:8.3μM
Kd: 300 nM
cat-ELCCA
11.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate 299
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Fluorescence Polarization (FP)
F
Acceptor
Fluorophore
Emission
+
Polarized Light Signal Detected
Depolarized
Light - No Signal
FRET Signal
D
No FRET Signal
Detected
F
RNA +
Fluorophore
+
RBP
No Inhibitor
+ Inhibitor
Fluorescence Resonance Energy Transfer (FRET)
A
RNA +
Acceptor
Fluorophore
+
+ Donor
Fluorophore
D
RBP
No Inhibitor
+ Inhibitor
F
Slow Rotation
+
Fast Rotation
D
A
A
Figure 11.10 Schematic representation of fluorescence polarization and fluorescence
resonance energy transfer assays to detect RPIs and their inhibition. Figure created in
BioRender.
Catalytic Enzyme Linked Click Chemistry Assay (cat-ELCCA)
HRP
RBP +
Biotin
+
+
RNA
5′-trans-
cyclooctene
SmBiT
mTet
TCO
RNA +
StreptavidinCoated Well
Split Enzyme Assay (SEA)
LgBiT
RBP
TCO
HaloTag
No Inhibitor
+ Inhibitor
No Inhibitor
+ Inhibitor
+
TCO
+
mTet
NanoBiT Enzyme
Click
+
mTet
Wash
Wash
NanoBiT
Substrate
Click
Click
Catalytic Chemiluminescent
Signal
No Signal
Chemiluminescent
Signal
NanoBiT
Substrate
No Signal
Figure 11.11 Schematic representation of catalytic enzyme linked click chemistry assay
and split enzyme assay to detect RPIs and their inhibition. Figure created in BioRender.
is immobilized onto a streptavidin-coated well plate and treated with an RNA
substrate harboring a trans-cyclooctene (TCO) click chemistry handle. Signal is
generated in this assay when the TCO-labeled RNA becomes covalently conjugated
to a methyltetrazine (mTet)-functionalized horseradish peroxidase (HRP) via
inverse-electron demand Diels–Alder-based click chemistry. Chemiluminescence
results after the addition of an HRP substrate. Key advantages of cat-ELCCA are
enhanced sensitivity due to catalytic signal amplication and minimal compound
interference compared to uorescence-based detection assays. Using this strategy,
a high-throughput screen was conducted against the pre-let-7-Lin28 RPI resulting
in the discovery of a novel Lin28 inhibitory scaold (Table 11.2) [176]. There
are, however, a couple of limitations to this approach including the use of costly
300 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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streptavidin-coated plates needed for protein immobilization and the multiple
washing steps required throughout the assay protocol. This ultimately led to the
development of a second click chemistry-based biorthogonal assay.
SEA is a protein complementation-based assay that utilizes a split luciferase that
is able to produce catalytic signal amplication following interaction between a target RNA and RBP [171]. The split NanoLuc Binary Technology (NanoBiT) system
from Promega is comprised of an 18 kDa larger subunit as well as an 11 amino acid
peptide making up the small subunit (SmBiT) [178]. These two components were
intentionally engineered to have low anity for each other with a Kdof 190 μM,
allowing for the components conjugated to these two subunits to be the main driver
of interaction [178]. Like cat-ELCCA, SEA also uses a TCO-labeled RNA. LgBiT is
fused to the RBP of interest, while SmBiT contains the mTet modication, which is
appended via conjugation to HaloTag [179]. Signal results when the RBP binds to
the target RNA following a covalent click chemistry reaction between the TCO and
mTet components, placing SmBiT and LgBiT in close enough proximity to assemble
the active NanoBiT enzyme which produces a chemiluminescent signal following
treatment with NanoLuc substrate. SEA has been utilized to detect RPIs in 384-well
plate format with robust assay statistics and was further able to detect inhibition by a
small molecule (CCG-233094 in Table 11.2) without the need to wash away unbound
species due to the low inherent anity of the NanoBiT subunits [171].
11.3.2.1 Cell-Based RPI Detection Assays
While enabling assay technologies, in vitro methods come with a major drawback
as they do not assess how RBPs behave in cells or how they may interact with RNAs
in cells due to the absence of post-translational modications or other proteinbinding partners present in the native cellular environment [169, 180, 181]. Indeed,
many RBPs function in complexes containing multiple RPIs or PPIs that facilitate
the interaction between the RNA and RBP [180]. This biological complexity
necessitates the need for cell-based methods to detect specic RPIs that are also
amenable to high-throughput experimentation and are reversible to allow for the
identication of inhibitors of the interaction. Cell-based platforms for detecting and
screening RPIs also come with an additional advantage in that they also allow for
simultaneous assessment of cellular permeability, activity, and cytotoxicity.
Cell-based FRET and uorescence in situ hybridization (FISH) coupled with
immunouorescence have broad applications and can be used to demonstrate
interaction and co-localization of an RNA and RBP [182–184]. However, FRET, in
addition to the limitations in uorescence-based methods already discussed, is also
highly dependent upon the ratios of the interacting partners to produce a signal.
Additionally, for these imaging-based methods to be made high-throughput, highly
specialized and expensive high-content imaging equipment is necessary. With FISH
coupled with immunouorescence specically,there is no evidence of a direct interaction between an RNA and protein, just co-localization of the components [185].
These methods also typically require cell xation and permeabilization to be used
with antibodies, making them not suitable for high-throughput experimentation.
Furthermore, only a fraction of RBPs have validated antibodies for use, making
11.3 Experimental Methods to Detect and Screen for Small Molecules that Modulate 301
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Trimolecular Fluorescence Complementation (TriFC) Assay RNA Fluorescent three-Hybrid (rF3H)
Ex. Em.
MS2CP
MS2 Hairpins RNA Target
Fluorescence Based RPI Detection in Cells
Reconstituted
Split
Fluorophore
RBP
Co-Localization
GFP
Lacl
Multiple lacO Clusters
MS2CP
MS2 Hairpin
RFP
RBP
RNA
Figure 11.12 Schematic representation of trimolecular fluorescence complementation
assay and RNA fluorescent three-hybrid assay to detect RPIs in a cell-based setting. Figure
created in BioRender.
these approaches not ideal or maybe even not an option for the detection of poorly
characterized RPIs [186].
Additional methods include the trimolecular uorescence complementation
(TriFC) assay and RNA uorescent three-hybrid (rF3H) assay (Figure 11.12).
TriFC, originally established for the visualization of cellular PPIs, utilizes a split
uorophore as the detection method, brought together by other interacting units
[187–190]. In this case, an RNA target sequence is appended to the sequence coding
for the MS2 hairpin RNA. The RNA bacteriophage MS2 coat protein (MS2CP),
which binds to the MS2 hairpin RNA, is fused to a portion of a split uorophore
[191]. The RBP-of-interest is fused to the second half of the split uorophore. When
the RBP binds to the RNA target adjacent to the MS2 hairpin RNA, the uorophore
is reconstituted and will exhibit a uorescence signal at its specic excitation and
emission wavelength, driven by the RPI. This assay has not been used for screening
inhibitors likely due to the irreversibility of the complementation between the uorophore protein fragments leading to high background and resulting in an inability
of this method to measure RPI dynamics [192]. In rF3H, an MS2 stem-loop-labeled
RNA (similar to that used in TriFC) is anchored to a specic locus by a fusion
protein containing the MS2CP, a GFP, and an anchoring protein. Specic anchor
proteins may direct the locus of the trap, with LacI binding to an integrated lab
operon shown as an example in Figure 11.12. Other loci include the Lamin B1
anchor localizing to the nuclear lamina or the Coilin protein localizing to Cajal
bodies [193]. Interaction of the RBP fused to another uorescent protein (RFP) with
the RNA target will give insight into co-localization of the protein to that specic
cellular compartment. This method, while not advantageous for screening, could
be useful for signal detection for certain disease contexts by enrichment of signal at
a specic locus.
11.3.3 Cell-Based RNA–Protein Interaction Screening
The limitations observed with in vitro small molecule screening, coupled with
the absence of cell-based screening methods for RPIs, led to the development of
additional cell-based RPI assays. RNA interaction with protein-mediated complementation assay (RiPCA; Figure 11.13), in similar manner to cat-ELCCA and SEA,
leverages biorthogonal chemistry strategies for the detection of specic interactions
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RNA-interaction with Protein-mediated Complementation Assay (RiPCA)
SmBiT-
Cytoplasm
HaloTag
Nucleus
RBP-
LgBiT
Transfect
RBP-
LgBiT
RBP
LgBiT
SmBiT
HaloTag
Transfect
No Inhibitor
+
Cl
+ Inhibitor
RNA + Chloroalkane
Cl
NanoBiT Enzyme
NanoBiT
Substrate
Chemiluminescent
NanoBiT
Substrate
Signal
No Signal
Figure 11.13 Schematic representation of RNA interaction with protein-mediated
complementation assay to detect RPIs in the presence of an inhibitor and without in a
cell-based setting. Figure created in BioRender.
[194–196]. The RBP-of-interest is fused to the LgBiT subunit, while the SmBiT
is fused to HaloTag (HT). Flp-In HEK293T cells are engineered to stably express
the SmBiT-HT fusion protein, which are subsequently transiently transfected
with a plasmid encoding the RBP fused to LgBiT. The RNA, functionalized with a
PEGylated chloroalkane substrate for conjugation to HT, is co-transfected alongside
the RBP-LgBiT. Signal is detected following HT covalently modifying the RNA
target and the RBP binding to the RNA, bringing LgBiT and SmBiT in proximity
to interact and form the catalytically active NanoLuc enzyme, which results in
the production of chemiluminescent signal following treatment with NanoLuc
substrate. Carried out in this way, a new cell line does not need to be generated for
each RBP- or RPI-of-interest, rather, a dierent RBP would just need to be cloned
into the plasmid-containing LgBiT. RiPCA is suitable for screening to identify
inhibitors of an RPI, is amenable to high-throughput experimentation, and results
in sensitive signal generated by the complex that is stabilized by the covalent bond
between HT and the RNA. Additionally, the cells are not lysed or permeabilized
to allow for signal detection because RiPCA uses Promega’s Live-Cell NanoBiT
detection reagent. Some limitations to this approach include potential issues with
overexpression of the RBP because it is transiently transfected through the plasmid
on top of endogenous expression. Moreover, the RNA must be generated through
chemical synthesis due to requirement of a site-specic nucleobase modication
for coupling to the chloroalkane handle for HT conjugation, limiting the length of
RNA that can be used due to limitations of RNA chemical synthesis.
11.4 Closing Remarks
Sequencing of the human genome catalyzed expanded exploration of RNA biology.
From these investigations, we have come to appreciate not only the complexity
and signicance of cellular RNAs but also how entangled the cellular lifecycle
of an RNA is with a cadre of regulatory proteins. As we continue to push the
boundaries of drug discovery toward the targeting of historically dicult-to-drug
areas of biology, RNAs and RPIs have emerged as exciting challenges for the eld
of medicinal chemistry. In learning more about RBPs and RPIs, we hope that new
insights will be gleaned with respect to molecular recognition principles for binding
References 303
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to RNAs that can be applied toward our pursuit of developing small molecule-based,
RNA-targeted therapeutics, as well as reveal new mechanisms for impacting RNA
biology in disease states for the development of next-generation medicines.
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