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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5402_Библиотеки_им_академика_М_И_Перельмана
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Figure 11.1 HuD RRM
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interacting with c-fos RNA.
Pink: β-sheet, green: α-helix,
gray: loop, and tan: RNA
(PDB: 1FXL).
11.1 Molecular Basis of RNA–Protein Interactions 285
the bases of the nucleotides and a third aromatic residue is often inserted between
two sugar rings of the dinucleotide (Figure 11.2) [41]. This β-sheet surface of the
RRM shows intrinsic preference for a particular RNA sequence. Structures of RRMs
bound to various RNAs have revealed a more complex mode of binding involving
interactions in the protein loops (as little as 1 or as many as 3) connecting the
β-sheets and α-helices. The loops primarily on the bottom end of the RRM structure
(loops 1, 3, and 5) have been shown to engage in RNA interactions in many proteins
(Figure 11.3), although exceptions have also been reported (e.g. NELF E) [48–50].
Interestingly, some loops have been shown to recognize a certain shape in an
RNA as opposed to sequence by binding to the phosphodiester backbone of the
major groove [48]. In some cases, when an aromatic residue is missing from the
RNP consensus sequence, interactions which utilize the loop portions become
the predominant mode of binding [51]. This loop-mediated binding mode was
hypothesized to have evolved later to expand the RNA recognition landscape of
RRMs [14]. As such, RRMs are often considered “plastic,” owing to their ability
to bind to a wide range of RNA targets, driven by their many modes of binding
and exible linker regions separating the domains, leading to their wide range of
functional activities [41].
286 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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β2
β3
β1
β4
Phe170
Figure 11.2 HuD RRM β1andβ3 aromatic residue stacking interaction with dinucleotide.
Pink: β-sheet, green: α-helix, gray: loop, and tan: RNA (PDB: 1FXL).
Loop 3
Figure 11.3 RRM-containing RBMY loop interaction with hairpin RNA. Pink: β-sheet,
green: α-helix, gray: loop, and tan: RNA. (PDB: 2FY1).
Tyr128
Loop 1 Loop 5
11.1.2 Double-Stranded RNA-Binding Domains (dsRBD)
Double-stranded RNA-binding domains (dsRBD), which are the second most
abundant RBD, represent a small protein domain (70 amino acids) that interacts
with double-stranded regions of RNA and contain a conserved α1β1β2β3α2topology
(Figure 11.4) [18]. Unlike other RBDs, dsRBDs typically recognize RNA secondary
structure rather than sequence [52]. dsRBDs primarily recognize A-form helices of
RNA, but can also bind to RNA hairpin structures [53, 54]. These proteins interact
with the RNA in both the minor and major grooves using three distinct regions
11.1 Molecular Basis of RNA–Protein Interactions 287
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Figure 11.4 Stau1 dsRBD
in complex with dsRNA.
Interaction through 2 minor
grooves and 1 major groove.
Pink: β-sheet, green: α-helix,
gray: loop, and tan: RNA.
(PDB: 6HTU).
from the protein to interact with three distinct regions on the RNA (Figure 11.4)
[54, 55]. Like many RBDs, dsRBDs are often found in multiple copies in an
RBP. A widespread auxiliary function of these domains is their role in regulating
nucleocytoplasmic transport of an RNA; however, they also function in RNA
interference, RNA processing, RNP and RNA localization, as well as RNA editing
and translational control [15, 56, 57].
11.1.3 Zinc Finger (ZnF) Domains
Zinc nger (ZnF) domains, more commonly thought of as DNA-binding transcription factors, also are involved in many RNA-binding events [16]. ZnFs can be found
alone in an RBP as tandem repeats or in combination with other RBDs [18]. These
domains are among the smallest of RBDs at 30 amino acids in length displaying a
conserved β1β2α1fold held together through coordination of a Zn2+ion (Figure 11.5)
[18]. There are many types of ZnFs including CCHH, CCCH, and CCCC, where C
represents cysteine and H is histidine, describing the amino acids in the ZnF domain
which coordinate to the Zn2+ion [18]. CCHH is the most common domain capable
of binding both DNA and RNA [58, 59]. Further evidence of RNA binding by ZnFs
came from structures of CCCH, CCHC, and CCCC ZnFs solved in complex with
single-stranded RNAs [31, 60–62]. ZnF recognition of an RNA sequence is based on
the type of ZnF domain present. For example, CCCH ZnF proteins typically bind to
AU-rich elements (AREs) located in the 3′untranslated region (3′UTR) of mRNAs
[63]. The primary mode of interaction between the protein and RNA is mediated
through hydrogen bonding of the protein backbone atoms with Watson–Crick edges
288 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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of the bases (Figure 11.5) [61]. This binding mode, however, is not standard for all
ZnF domains. A unique binding mechanism is observed, for example, with MBNL1
whose crystal structure demonstrated that it binds to 5′GCU 3′sequences with its
ZnF3 and ZnF4 domains [31]. In addition to hydrogen bonding interactions with the
protein backbone, this RPI is mediated by stacking interactions and hydrogen bonds
involving main chain atoms of the protein (Figure 11.6). Much of the structural data
available for ZnF domains has provided insights into the possible functions of these
2.54Å
1.92Å
1.87Å
2.44Å
Figure 11.5 Mbnl1 ZnF 1 and 2 in complex with cardiac troponin T mRNA. Pink: β-sheet,
green: α-helix, orange: Watson–Crick edges of base and hydrogen bonds (with distance)
gray: loop, and tan: RNA. (PDB: 5U9B).
Figure 11.6 Mbnl1 ZnF 4 stacking with RNA. Pink: β-sheet, green: α-helix, gray: loop, and
tan: RNA. (PDB: 3D2S).
11.1 Molecular Basis of RNA–Protein Interactions 289
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protein as alternative splicing regulators [31, 64]. However, due to the wide variety of
interactions possible through ZnF domains binding, more structural information is
needed to fully understand their diverse binding modes, and ultimately the specic
function(s) of these small RBDs [18].
11.1.4 K Homology (KH) Domains
K Homology (KH) domains are ∼70 amino acids in length and separated into two
categories: type I in eukaryotes and type II in prokaryotes, each of which exhibits
dierent folds. The topology of the type I KH domain is described as β1α1α2β2β3α3,
while the reverse is true of the type II fold that contains an α1β1β2α2α3β3topology
(Figure 11.7a) [17, 65]. The binding surface of a KH domain forms a small crevice
through the α1 and α2 helices on one side and the β1 sheet on the other. This
Figure 11.7 (a) Poly(c) binding
protein KH interaction with 4
nucleotide region of RNA. (PDB:
2PY9) (b) NusA tandem KH
interacting with 11 nucleotide
region of RNA. Pink: β-sheet, green:
α-helix, grey: loop, and tan: RNA.
(PDB: 2ATW).
(a)
(b)
290 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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small pocket cannot accommodate a large structure; thus, RNA recognition by a
KH domain is accomplished through only four residues [17]. Nonetheless, KH
domains can still interact with many dierent combinations of four nucleobases
(Figure 11.7a) [18]. Binding to a particular four-base sequence on its own yields
relatively low micromolar anity; however [18], when multiple KH domains are
present, as seen with the other RBDs, there is a cooperative eect resulting in
higher binding anities. Using NusA as an example, this protein contains two KH
domains separated by a short linker allowing for numerous contacts between the
domains and forms a larger plane of interaction on the RNA resulting in nanomolar
binding anity (Figure 11.7b) [66].
11.1.5 Other RBDs
The other RBDs include cold shock domains (CSD), YT521-B homology domains
(YTH), RGG/RG domains, DEAD/DEAH box helicase domains, Pumilio (PUF)
domains, Piwi/Argonaute/Zwille (PAZ) domains, and Sm domains. CSDs are
comprised of ∼70 amino acids and, similar to RRMs, contains an RNP1 and RNP2
motif [67]. This domain, more commonly found in eukaryotes, contains a β-barrel
structure composed of ve antiparallel β-sheets [67]. The YTH domain, roughly
100–150 amino acids in length, is part of a family of proteins, which function as
readers of 6-methyladenosine (m6A) modications, which are the most common
internal modication found on RNAs [68]. Binding of YTH-containing RBPs to
m6A modications subsequently results in either a decrease in RNA stability and
degradation through recruitment of the CCR4-NOT complex [69] or regulation of
translation eciency through interactions with the translation initiation machinery
and the ribosome [70]. RGG/RG motifs are dened by sequences rich in arginine
residues, which are positively charged and capable of mediating hydrogen bonding
and amino-aromatic interactions [71]. This motif is found in more than 1000 proteins and has important functions in transcription, splicing, DNA damage signaling,
and mRNA translation through binding to RNA and DNA [72–77]. DEAD/DEAH
box helicase domains are responsible for unwinding DNA and dsRNA [78]. PUF
domains are a large protein domain consisting of eight α-helices, which repeat a
highly conserved sequence of 36 amino acids [79, 80]. Some functions of the PUF
proteins include regulation of RNA decay and translational repression [81]. PAZ
and Sm domains are mostly involved in microRNA (miRNA) binding and small
nuclear RNA (snRNA) binding, respectively [10].
11.2 Regulation and Dysregulation of RNA–Protein
Interactions
RBP regulation of RNAs is carried out at every step in the lifecycle of an RNA, and as
highlighted above, RNAs are invariablybound by both specic and nonspecic RBPs
from transcription to translation as depicted in Figure 11.8 [82]. As a model type of
RNA by which to exemplify the impact of RBPs on an RNA’s lifespan, we will use
Transcription
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11.2 Regulation and Dysregulation of RNA–Protein Interactions 291
Nucleus
Cytoplasm
Pre-mRNA
5' UTR
5'
Capping Enzyme
Pre-mRNA Processing
RBP RBP
Spliceosome
Introns
Exons
3' UTR
CPSF
Cleavage &
Polyadenylation
3'
RBP Regulates
Alternative Splicing
Mature mRNA
5' UTR
5'
G
5'
m7G
Coding
Coding
Nuclear
Export
RBP
RBP
3' UTR5' UTR
3' UTR
AAAAAA
RNA
Localization
AA A A
3'
RBP Stabilizes mRNA RBP Destabilizes mRNA
5'
G
Coding
3' UTR5' UTR
A A A
3'
3'
Enhances mRNA Translation to Protein Deadenylation & Degradation of mRNA
Figure 11.8 Representation of cellular functions of RBPs acting on mRNA targets from
transcription through pre-mRNA processing to translation. Figure created in BioRender.
mRNAs, which not only contain the code or “message” to make a protein but also
contain sites which provide information for regulation of its nuclear export, subcellular localization, translation, and stability by RBPs [13, 82]. Weencourage readersto
consults previous reviews to learn more about RBP regulation of non-coding RNAs
[83–85], as well as RNA-modifying enzymes [86, 87].
292 11 RNA–Protein Interactions: A New Approach for Drugging RNA Biology
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An mRNA is rst transcribed from DNA as a pre-mRNA, which then undergoes
splicing to form a mature coding mRNA, carried out by an RNP complex known
as the spliceosome. During pre-mRNA processing, an RNA will also undergo modication: capping at the 5′end and polyadenylation of the 3′end of the mRNA.
These modications are read by RBPs important in regulating the translation of an
mRNA. For example, the 5′cap is bound by eIF4E, which controls the initiation
of cap-dependent translation, and the poly A tail of the mRNA is regulated by poly
A binding proteins to similarly facilitate translation as well as decay. In addition to
the coding sequence, and outside of these terminal modications, a mature mRNA
also contains untranslated regions at both the 5′and 3′end (5′UTR and 3′UTR,
respectively). RNA regulation by RBPs mainly occurs through interactions at these
noncoding regions, but in some cases, can also occur in the coding region [88–91].
The 5′UTR contains various cis-acting regulatory RNA elements and structures
which serve roles in the recruitment of various RBPs, ribosome entry,and interaction
with non-coding RNAs [92]. These sites within the 5′UTR are mainly important for
regulation of translation initiation [93]. The 3′UTR of mRNAs serves as a hotspot
for RBP binding and is important for regulation of translation initiation, as well as
controlling every other aspect of mRNA fate such as subcellular localization, mRNA
stability, and polyadenylation [93, 94]. Additionally, the 3′UTR contains sites for the
binding of miRNAs [94].
As RBPs play important roles in all cellular processes, deviations from the tightly
controlled regulation of RBPs and RPIs are the basis for many diseases. In many
cases, mutations in genes encoding for RBPs have emerged as critical determinants
of neurological disorders such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), multisystem proteinopathy (MSP), and frontotemporal lobar
degeneration (FTLD) [95]. Further, the 3′UTR of many proto-oncogenes, tumor
suppressor proteins, inammatory cytokines, and growth factors are enriched with
AREs, meaning their expression levels are tightly regulated by the binding of various
RBPs, which interact with those elements [96]. A recent review describes many
examples in which dysregulation of RBPs is associated with cancer chemoresistance
providing many excellent examples [97]. Here we describe several mechanisms
through which disruptions in the vast network of RPIs may alter normal cell
function and result in various diseases (depicted in Figure 11.9). The examples will
include a discussion of RBPs, which also contain each of the four main types of
RBDs presented in Sections 11.1.1–11.1.4.
11.2.1 Poor Quality Control Leads to Over- and Underproduction
of RBPs
Aberrant expression of RBPs causing upregulation or downregulation of an individual protein contributes to disease onset. This is the case for the RRM-containing
proteins heterogenous ribonucleoprotein A/B (hnRNP A/B) and Musashi 1/2
(Msi1/2). Depleted levels of the alternative splicing regulators hnRNP A/B, suppressed directly by impaired cholinergic signaling, cause key disease hallmarks in
Alzheimer’s disease [98]. At the same time, hnRNP A1 overexpression has also been
11.2 Regulation and Dysregulation of RNA–Protein Interactions 293
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1. Aberrant
Expression of RBPs
Pre-mRNA
Mature-mRNA
5. Dysregulation
in Post-
transcriptional
Gene Expression
m7G
5'
Stability Translation Decay
Activation of Cancerstimulated Pathways
Proliferation
Transcription
5'
m7G
5'
Nucleus
Pre-mRNA Processing
RBP
RBP
Spliceosome
3. Dysregulated
Nuclear Export
AAA A
RBP
AngiogenesisInvasion
2. Dysregulation of
RNA Processing
RBP
3'
A A A
5'
3'
A
3'
5'
m7G
m7G
5'
5'
m7G
Cytoplasm
6. RNP Complex
Membrane-less
Organelle Formation &
RPI Aggregation
m7G
RBP
AAA A
3'
RBP
m7G
AAA A
RBP
m7G
5'
AAA A
RBP
Neurodegenerative
Disease
RBP
4. Aberrant
Localization of RBPs
AAA A
3'
AAA A
3'
RBP
3'
AAA A
3'
RBP
3'
Figure 11.9 Representation of several ways in which RBPs and RPIs can be dysregulated
in cells contributing to two main disease areas: cancer and neurodegeneration. Figure
created in BioRender.
linked to induced alternative splicing of the amyloid precursor protein mRNA and
reduced Aβ levels, further suggesting that hnRNP A/B reduction could contribute
to Alzheimer’s Disease onset [99]. Msi1/2 overexpression, on the other hand, is
found to contribute to almost all hematological malignancies and is correlative
with poor clinical prognosis in these malignancies [100]. Msi1/2 play distinct roles
in the development and maintenance of neural cells with high levels of transcripts
present in the brain during postnatal development, low expression persisting
into adulthood, and a resurgence of expression in various malignancies [101].
This family of proteins contains tandem RRM domains and bind the sequence
motif 5′UAGUAG 3′in the 3′UTR of target RNAs [102]. Msi1/2 have emerged
as biomarkers and therapeutic targets. One interaction with which Msi1/2 are
hypothesized to contribute to disease pathogenesis is by binding to and repressing
the translation of the Numb1 mRNA, resulting in increased NOTCH1 signaling in
cancer and leukemia patients [23]. This mRNA encodes for the Numb1 protein,
which is involved in controlling cell fate decisions in the central and peripheral
nervous system through inhibition of NOTCH1 [103].
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11.2.2 RBPs Become Out of Control, mRNA Processing Gets a
Makeover (and Hates It)
In addition to 5′capping and 3′cleavage/polyadenylation, splicing of pre-mRNAs is
a vital step in dictating gene expression, especially in eukaryotes where many proteins are produced from a single gene locus [104]. This process is controlled by an
RNP complex containing the core spliceosome, accessory RBPs, and RNA. This is
a major point of dysregulation in the cell arising from the fact that RBP–mRNA
interactions are transient and exhibit relatively low specicity [105]. Here we will
focus on the auxiliary RBPs that function in splicing regulation but do not make
up the core spliceosome. There are three ways in which spliceopathy is induced in
cells in an RBP-dependent manner: (i) disruption of a splicing element, (ii) mutations aecting the activity of RBP splicing factors, and (iii) toxic RNA (example to be
discussed in Section 11.2.3) [105]. Disruptions in a splicing element refer to a mutation causing a change in splice site recognition aecting recognition by trans-acting
RBPs. The SR- and hnRNP RRM-containing protein families are principle splicing factors that bind auxiliary enhancer and silencer sequences in the pre-mRNA
and act as activators and repressors of splicing in a context-dependent manner contingent upon binding to an intron or an exon [106, 107]. Mutations aecting the
activity of RBPs can result in up/downregulation of protein levels and aect splicing
in a similar manner as observed with the hnRNP A/B example in Section 11.2.1. In
the case of serine arginine-rich splicing factor 1 (Srsf1), depletion leads to aberrant
expression of its primary target BIN1, leading to reduced ability to repress c-Myc
[108]. Other targets whose splicing is regulated by Srsf1 include those that promote
proto-oncogenic transformation [109]. Major splicing regulators are essential to normal cellular function due to the signicance of the mRNAs they produce, as well as
the fact that many of these regulators are responsible for the alternative splicing of
hundreds of transcripts, demonstrating the multitude of processes aected by a single RBP [110–112]. Further information about the eects of splicing dysregulation
in cancer can be found in a recent review [113].
11.2.3 RBP Shuttling of mRNA Becomes Askew
Mature mRNAs are immediately exported from the nucleus to the cytoplasm
after processing is complete. Many proteins are involved in making sure RNAs
are exported properly from the nucleus to the cytoplasm and further to the site of
translation including the nuclear pore complex and other RBPs. This is a vital step
in the regulation of gene expression in which aberrantly transcribed or processed
mRNAs are not shuttled from the nucleus, as well as the fact that this step is highly
controlled by extracellular signaling and stress responses [114]. At the same time,
however, nuclear retention of RNA may also contribute to dysregulation and is the
basis for several diseases [115]. One of the most well-characterized examples of this
is the mutation causing the multisystemic disorder, myotonic dystrophy (DM1),
and other repeat disorders [116, 117]. DM1 is caused by a CTG expanded repeat in
the Dystrophia Myotonica Protein Kinase (DMPK) gene resulting in mutant mRNA
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