Available online at www.sciencedirect.com
Alternative splicing of intrinsical
ly disordered regions andrewiring of protein interactionsMarija Buljan1, Guilhem Chalancon1, A Keith Dunker2, Alex Bateman3,S Balaji1, Monika Fuxreiter4 and M Madan Babu1Alternatively spliced protein segments tend to be intrinsically
disordered and contain linear interaction motifs and/or post-
translational modification sites. An emerging concept is that
differential inclusion of such disordered segments can mediate
new protein interactions, and hence change the context in which
the biochemical or molecular functions are carried out by the
protein. Since genes with disordered regions are enriched in
regulatory and signaling functions, the resulting protein isoforms
could alter their function in different tissues and organisms by
rewiring interaction networks through the recruitment of distinct
interaction partners via the alternatively spliced disordered
segments. In this manner, the alternative splicing of mRNA coding
for disordered segments may contribute to the emergence of new
traits during evolution, development and disease.
Addresses1 MRC Laboratory of Molecular Biology, Francis Crick Avenue,
Cambridge CB2 0QH, United Kingdom2 Center for Computational Biology and Bioinformatics, Department of
Biochemistry and Molecular Biology, Indiana University School of
Medicine, Indianapolis, IN 46202, USA3 European Bioinformatics Institute, Wellcome Trust Genome Campus,
Hinxton, Saffron Walden CB10 1SD, United Kingdom4 DE OEC-Momentum Laboratory of Protein Dynamics, University of
Debrecen, H-4032 Debrecen, Nagyerdei krt 98, Hungary
Corresponding authors: Buljan, Marija ([email protected])
and Babu, M Madan ([email protected])
Current Opinion in Structural Biology 2013, 23:443–450
This review comes from a themed issue on Sequences and topology
Edited by Julian Gough and A Keith Dunker
For a complete overview see the Issue and the Editorial
Available online 22nd May 2013
0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.sbi.2013.03.006
IntroductionOne of the major challenges in biology is to understand
how novel phenotypic traits emerge and to describe their
underlying molecular mechanisms. Numerous studies
have highlighted the importance of gene duplication
and recombination on one side [1,2] and alterations in gene
expression regulation on the other side [3] as fundamental
for the evolution of new phenotypic traits. Recently,
alternative splicing (AS) has been shown to contribute
significantly to changes in proteome composition between
organisms and to the increase in cellular complexity and
diversity of phenotypes among eukaryotes [4,5��,6��,7].
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While AS plays a key role in the expansion of proteomic
and regulatory complexity, the molecular and biochemical
functions of differentially spliced exons remain largely
uncharacterized [8]. AS tends to avoid protein domains
but more often affect intrinsically disordered (ID) regions
[9–12]. However, the general principles behind how these
different isoforms contribute to the functional versatility
and how they mediate the emergence of novel phenotypes
is just beginning to be grasped [5��,6��,13��,14��]. ID
regions are polypeptide segments that do not acquire a
defined tertiary structure autonomously but adopt diverse
interconverting conformational states [15–18]. Frequently,
disordered regions fold completely upon binding [19]
while in some cases such regions remain disordered even
in the bound state [20,21]. Here, we synthesize emerging
concepts to shed light on how AS of disordered regions
might rewire molecular interactions, and how this may
facilitate the emergence of novel phenotypes. In particular,
we discuss how such events could be crucial for defining
tissue-specific (TS) and organism-specific traits. While AS
of structured domains may lead to similar effects, these are
not discussed here.
How do disordered regions increase the functional
versatility of proteins?
ID segments increase the functional versatility of proteins
by: (a) providing conformational heterogeneity between
structured domains, thus increasing the number of confor-
mational states (Figure 1a); (b) presenting linear peptide
motifs [22] that bind to other proteins, nucleic acids and
small molecule ligands, thereby enabling larger protein
interaction landscapes (Figure 1b) and (c) presenting
amino acids that become post-translationally modified
(PTM), thereby altering the chemical nature of the poly-
peptide and further increasing its diversity of interactions
(Figure 1c). In addition, the overall net charge of the
disordered segment, the modification state, and the amino
acid composition can all influence the conformational
ensemble and the chemical states of the entire protein,
thereby altering the functions of the full-length proteins
[18,23–32].
Splicing of disordered segments rewiresinteraction networksSpliced ID regions often harbor functionally important
sites
Earlier computational studies of alternative protein iso-
forms from the UniProt database (http://www.uniprot.org/)
as well as structures of different isoforms in the PDB
Current Opinion in Structural Biology 2013, 23:443–450
444 Sequences and topology
Figure 1
Embed post-translational modification sites
Increases conformational heterogeneity Tunes interaction specificity, affinity & avidity
Disordered segments increase the functional versatility of proteins
Connect structured domains together Contain linear peptide motifs
Alters chemical composition and regulates interaction properties
P
Current Opinion in Structural Biology
Disordered segments increase the functional versatility of proteins. Disordered segments are represented in white, and structured domains as gray
and blue blobs. A disordered segment can influence the functional versatility of a protein by increasing the conformational heterogeneity between
domains and the number of conformational states achieved by the protein (left panel). Linear peptide motifs (green rectangles) embedded in
disordered segments can tune the specificity, selectivity, kinetics, affinity and avidity of interactions with distinct partners (blue blobs) (middle panel).
Post-translational modification sites embedded in disordered segments can modify the function by altering the segment’s chemical composition and/
or by serving as regulators of diverse interaction properties (right panel).
(http://www.rcsb.org/) suggest that AS exons in mRNA
often code for ID regions [9,12]. Calculations on a much
larger set of 15 678 proteins with 36 320 alternatively
spliced regions confirms these observations and suggest
that a higher portion of alternatively spliced mRNA codes
for ID regions as compared to that which codes for struc-
tured regions (Dunker et al., unpublished results). AS protein
segments in general [33��] and tissue-specific AS segments
in particular [13��,14��] frequently contain disordered
regions with embedded linear interaction motifs. This
suggests that the alternative inclusion of such functional
elements can trigger changes in protein function by alter-
ing its stability, subcellular location or by influencing its
molecular interactions [13��,14��,33��]. For example, the
alternative inclusion of peptide KEN-motifs affect protein
half-life by targeting specific isoforms for degradation, the
alternative inclusion of NLS signal peptides affect the
subcellular location of proteins, or the alternative inclusion
of motifs that bind PDZ, PTB, SH2, SH3 or WW domains
modulate interactions with these signaling domains
[13��,33��,34,35].
AS disordered regions can also display the following charac-
teristics: the segment (a) lacks linear interaction motifs or
PTM sites [12,13��], thus inclusion of such segments may
increase the conformational entropy or may alter the bind-
ing affinity of the full-length protein due to multiple weak
transient interactions [20,21]; (b) contains multiple occur-
rences of the same motif [33��], thus altering the avidity of
an interaction [36] or increasing the local concentration of
Current Opinion in Structural Biology 2013, 23:443–450
an interacting protein within specific regions in a cell [37];
(c) contains distinct, possibly overlapping, motifs or Mol-
ecular Recognition Features (MoRFs) [38�], thus confer-
ring the property of an interaction hub, and thereby
contributing to multiple functional outcomes, including
moonlighting [39]; and (d) contains multiple occurrences of
PTM sites [13��], thus changing the overall charge distri-
bution and influencing the conformational states and fine-
tuning the interactions with other molecules [25,27] in-
cluding the alteration of partner selection preferences
[38�].
Tissue-specific rewiring of interactions through spliced
ID segments
Buljan et al. [13��] recently analyzed published transcrip-
tome data from several different human tissues and
cancer cell lines [40] and found that tissue-specific AS
protein segments frequently contain ID regions that
embed functional elements such as evolutionarily con-
served interaction motifs and PTM sites. Furthermore,
genes with TS exons tend to occupy central positions in
interaction networks and tend to display distinct inter-
action partners in the respective tissues. Many of these
genes have roles in signaling and development and are
associated with cancer and other human diseases. In an
independent study, Ellis et al. [14��] reported similar
findings, and, using an automated protein–protein inter-
action assay, they demonstrated a direct connection be-
tween the AS disordered segments and protein binding
properties. Specifically, approximately one-third of the
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Rewiring protein interactions by splicing ID regions Buljan et al. 445
analyzed neural-regulated exons were observed to affect
protein–protein interactions. These two studies establish
the concept that AS of ID segments with embedded
functional elements can rewire protein interactions, regu-
latory networks and signaling pathways in a tissue-specific
manner, thus conferring functional versatility to proteins
and increasing interaction network diversity across tissues
[13��] (Figures 2 and 3).
Not only disordered regions but also structured domains
can undergo AS. AS affecting structured domains have
been observed to rewire biomolecular interactions
[41�,42��,43,44]. For instance, an evolutionarily conserved
Figure 2
D1
D2
NT
AB
Acid Box linker(inhibition module)
FGF ReceptorReceptorttFGFFGF Receptor
PEX3
Binary intera
AAAA
DNA
mRNA
Protein
Molecular mechanisms by which spliced disordered segmen
(a) (b)Auto-inhibitory interaction
Molecular mechanisms by which alternatively spliced ID segments influence
can regulate protein interactions via distinct mechanisms. (a) ASDS can infl
masking or competing for the interaction interface via auto-inhibition. For ex
Factor (FGF) receptor prevents the protein from interacting with its ligand F
segment connects the immunoglobulin domains D1 and D2 (cyan, surface a
can directly affect binary interactions, as shown for the binding of the human
(red helix within a disordered segment) (PDB:3MK4). (c) ASDS can influence
dependent protein kinase II (CAMKII) forms a dodecameric complex. Each h
and a C-terminal hub domain. These two domains are connected by a diso
Variation in the linker length directly impacts the sensitivity of CAMKII for ca
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embryonic stem cell specific AS of mutually exclusive
exons changes the DNA-binding preference of the fork-
head transcription factor FOXP1. The altered residues in
FOXP1’s DNA-binding domain affect specificity of DNA
binding and hence influence pluripotency and differen-
tiation of Embryonic Stem cells [42��].
Molecular mechanism of interaction rewiringvia splicing of disordered segmentsSplicing of disordered segments can affect a number of
properties of protein interactions. Below, we discuss how
such AS events influence intra-molecular and inter-mol-
ecular interactions [13��,45]. Note that through similar
PEX19 motif
Protein complex
NT kinasedomain
CT hubdomain
Ca2+/CaMbinding region
regulatorysegment
linker
(β)CAMK II(β)CAMK II
ction
AAAA
ts influence protein interactions
(c)
Alternatively spliced exon
Disordered segment
Structured domain
Constitutively expressed exon
Current Opinion in Structural Biology
protein interactions. Alternative splicing of disordered segments (ASDS)
uence the interaction of full-length proteins with other biomolecules by
ample, a segment with the Acid Box (AB; red oval) in Fibroblast Growth
GF1 and is subjected to alternative splicing. In the long isoform, this
nd cartoon representation; D1:PDB:2CKN and D2:PDB:1RY7). (b) ASDS
peroxin PEX3 (blue, surface representation) with the PEX19 binding motif
dynamics and kinetics of protein complexes. The Ca2+/calmodulin-
oloenzyme subunit (in cyan) comprises of an N-terminal kinase domain
rdered linker (red), the length of which varies among splice isoforms.
lcium gradients (PDB:3BHH).
Current Opinion in Structural Biology 2013, 23:443–450
446 Sequences and topology
Figure 3
Rewiring of protein-protein interaction networks
11 2 3 4
1 2 3 4
1 2 3 4
1 2 3 4
Mutations indisordered regions
Evolution ofsplicing patterns
Impact of mutations and splicing of disordered segments on disease and the evolution of new phenotypes
(across organisms or tissues)
(across organisms or somatic mutations)
Evolution of organism-specific and tissue-specific traitsand manifestation of disease
Increase in interaction network diversityacross tissues and organisms
Across tissues
Across organisms
differential usageof segments
that influence interactions
modificationof segments
that influence interactions
(a) (b)
Current Opinion in Structural Biology
Impact of mutations and splicing of ID segments on disease and the evolution of new phenotypes. (a) Alternative splicing of disordered segments can
cause the rewiring of protein interaction networks across different tissues and organisms. (b) Mutations in disordered regions, either across organisms
or during the lifetime of an organism (e.g. somatic mutations), can modify segments that influence protein interactions leading to new phenotypes or
diseases (e.g. cancer). Likewise, the evolution of new splicing patterns, and their tissue-specific regulation, permits the differential usage of segments
that influence interactions. The combination of both mechanisms not only permits evolution of organism-specific or tissue-specific traits, but can also
be involved in disease development.
principles, AS of disordered segments could also influ-
ence protein–DNA, protein–RNA and protein–small
molecule interaction networks.
Auto-inhibitory interactions
Auto-inhibition modules (IMs) influence interaction of
full-length proteins with other biomolecules by masking
or competing for the interaction interface (Figure 2a).
Trudeau et al. [46��] analyzed auto-inhibited proteins and
demonstrated that such IMs are enriched in intrinsic
disorder. By comparing the properties of proteins with
disordered IMs to those with structured IMs, they show
that ID IMs permit fine-tuning of the equilibrium be-
tween the active and inactive states. The disordered IMs
were observed to be more highly phosphorylated, more
frequently AS, and to contain evolutionarily conserved
linear peptide motifs that often changed their secondary
structure during activation [46��]. Such IMs may not only
be regulated by splicing but may also emerge indepen-
dently in organisms during the course of evolution. For
instance, an interaction between Homeobox HoxA11 and
Forkhead box 01A (Foxo1a) in mammals has evolved only
after a previously present, but masked, binding site in
Current Opinion in Structural Biology 2013, 23:443–450
HoxA11 became exposed through mutations in a disor-
dered region that mediated self-interaction [47]. Thus,
inclusion or skipping of such segments may contribute to
selectivity and altered kinetics in partner recognition.
Such properties may be further modulated through TS
splicing and hence contribute to fine-tuning of molecular
interactions in a spatially and temporally regulated man-
ner [13��].
Binary interactions
Linear motifs, MoRFs and PTMs within ID regions
enable molecular interactions. Thus the AS of ID seg-
ments containing these elements can form new molecu-
lar interactions and thereby lead to the recruitment of the
same biochemical or molecular functions (often
mediated by structured domains in the same polypep-
tide) into a different context (Figure 2b) [13��]. For
instance, differential inclusion of disordered segments
with interaction motifs in a kinase protein could cause the
resulting kinase to recruit completely different protein
substrates. Similarly, the differential inclusion of disor-
dered PTM sites (e.g. phosphorylation sites) could alter
whether a given protein becomes the substrate for a
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Rewiring protein interactions by splicing ID regions Buljan et al. 447
modifying enzyme (e.g. kinase). Thus AS of such dis-
ordered regions has the potential to rewire molecular
interaction networks and alter the specificity, selectivity,
affinity and kinetics of such molecular interactions [13��].Indeed, Merkin et al. [6��] recently investigated AS in
equivalent tissues across diverse organisms and found
that AS often alters whether a protein can be phosphory-
lated, thereby delimiting the scope of kinase signaling.
Similarly, changing the length of a disordered region can
interfere allosterically with interface formation else-
where due to increased conformational entropy
[48,49]. For example, alternative isoforms of the homo-
meric transcription factor Ulthrabithorax (UBX) gene,
which are expressed in a developmental and in a TS
manner, differ only in the length of the disordered
segment adjacent to the DNA-binding homeodomain
[48,50–52]. This AS region in UBX is not directly
involved in binding, but rather modulates the protein’s
dynamics and determines its DNA binding affinity. Thus
AS of the ID region enables UBX isoforms to recognize
different DNA sequences in distinct contexts and to bind
to the same DNA sequence differently. Hence, alterna-
tive inclusion of ID regions can have a significant influ-
ence on the affinity and kinetics of protein interactions
[13��].
Protein complexes
Many proteins form stable homo-oligomeric or hetero-
oligomeric protein complexes in the cell [53,54]. ID
regions in the interaction interface and their PTM often
influence complex formation [55,56]. Expression of the
subunit isoforms with differentially included ID seg-
ments can influence complex formation and cause tun-
able protein properties [57] (Figure 2c). Moreover, the
simultaneous expression of multiple isoforms that form
homo-complexes or hetero-complexes may lead to the
sequestration of functional isoforms into ‘heterogeneous’
oligomeric complexes, thereby leading to transient loss or
gain of function [58]. Depending on the expression level
of the isoform that encodes TS segments, the equilibrium
between the different oligomeric ‘protein-complex
states’ will be influenced, thereby leading to competition
for a common interaction partner [59] or altered kinetics
in response to a signal. This may result in dominant
negative effects or ultra-sensitivity due to molecular
titration effects [60,61]. For example, the increased diver-
sity of cell signaling options caused by receptor dimeriza-
tion is further enhanced by the generation of splice
variants in the case of GPCRs, one of the largest family
of membrane signaling proteins [62].
Implications for evolution, development anddiseaseEvolution and development
During evolution, ID regions contribute more extensively
than structured regions to the rewiring of protein inter-
action networks [63–65] by means of short insertions/
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deletions or point mutations that modulate interactions
(Figure 3). Fine-tuning of gained or rewired interactions
can also result from mutations of flanking residues around
an interaction motif [66,67]. Furthermore, species-
specific evolution of AS [68] of such altered disordered
regions may further expand the functional repertoire of
orthologous proteins by rewiring protein interaction net-
works in space (e.g. across tissues) or time (e.g. during
development or evolution) [68]. Indeed, the work of
Merkin et al. [6��] and Barbosa-Morais et al. [5��] revealed
the following trends: (a) AS is well conserved only for a
subset of exons and is frequently lineage-specific; (b) TS
spliced segments are enriched in PTM sites and disor-
dered regions; (c) tissue-specific AS often alters protein
phosphorylation sites, delimiting the scope of kinase
signaling; (d) certain splicing events likely remodel
protein interactions involving orthologous genes in equiv-
alent tissues across different organisms; and (e) segments
spliced in such species-specific manner are enriched in ID
regions and frequently occur in regulatory proteins.
Taken together, these studies suggest that diversification
of splicing and sequence mutations in ID regions during
the course of evolution may underlie the emergence of
novel phenotypic traits. These could include both tissue
differentiation and differences among the extant organ-
isms [5��,6��,7,13��] (Figure 3). We speculate that similar
principles might underlie the growing evidence for popu-
lation-specific [69–71] or sex-specific traits within a
species [72].
Disease
While molecular interactions can evolve through a small
number of mutations within ID segments due to the
emergence or loss of binding motifs, this property also
raises the risk of rewiring interactions that lead to
cancer or genetic diseases [73,74] (Figure 3). Indeed,
TS exons are significantly enriched in genes associated
with cancer and embryonic lethality [13��], thus under-
scoring the importance of TS exons in disease devel-
opment and the associated regulatory pathways. Since
TS isoforms likely have distinct molecular interactions,
this may explain how the same mutation may lead to
different phenotypes across tissue or organ types even
when gene expression levels across the tissues are
similar. Such tissue-specific AS-dependent gain or lo-
ss of specific interactions could lead to selective mani-
festation of disease in particular organs or tissue
types [74–76]. The flood of mutation data from the
cancer genome sequencing projects can be better inter-
preted by bearing in mind, not only which genes are
expressed but also which isoforms are expressed in the
tissue of interest. Exciting insights are already being
provided into how mutations that affect splicing events
or cause altered expression of certain isoforms — thus
leading to mis-regulation of protein function — can
cause diseases, such as cancer, in certain tissue types
[77,78].
Current Opinion in Structural Biology 2013, 23:443–450
448 Sequences and topology
Conclusion and future directionsRapidly advancing technology makes it possible to obtain
sequences and expression patterns of different isoforms
in diverse tissue types, disease states and different devel-
opmental stages. Thus, some of the major challenges are
to understand how sequence variation and differential
inclusion of exons leads to the rewiring of molecular
interactions at the genomic and molecular level, and then
to predict when these alterations affect phenotype or
cause disease. While interpreting the impact of differen-
tial inclusion of structured domains is more feasible,
understanding the impact of addition or removal of ID
segments remains a challenge. Hence, an important
direction for future research is to develop a comprehen-
sive characterization and categorization of PTM and
molecular interaction sites in ID regions and how the
former alters the latter. These improvements will involve
development and expansion of databases such as ELM
[79], PhosphoELM [80], iELM [81], and improved pre-
dictions of sites for PTMs and molecular interactions.
This knowledge will provide the molecular basis for
interpreting how and when the differential inclusion of
disordered segments can contribute to the generation of
novel phenotypic traits in molecular terms. Such
improved understanding could lead to therapeutic strat-
egies tailored to altering the functions of specific exons,
for example by developing small molecules that target AS
disordered segments (e.g. via antisense oligonucleotides
or targeted delivery of small RNA or small molecules in
specific tissues) while avoiding constitutive exons present
in all isoforms [58,82–86]. Finally, as sequencing individ-
ual human genomes at affordable cost is becoming a
reality, a better understanding of how natural variation
influences disordered regions, AS patterns and the rewir-
ing of molecular interaction networks will have a signifi-
cant impact for personalized medicine and for bettering
human health.
AcknowledgementsThis work was supported by the Medical Research Council (U105185859;M.B., G.C., M.F., S.B., and M.M.B.), the Wellcome Trust (A.B.), HFSP(RGY0073/2010; M.B. and M.M.B.), the EMBO Young Investigator Program(M.M.B.), ERASysBio+ (GRAPPLE; M.M.B.), the Gates CambridgeScholarship and the Knox Trinity Studentship (G.C.) and the Momentumprogram (LP2012-41) of the Hungarian Academy of Sciences (M.F.).
Note added in proofThe reader is referred to two relevant articles that were published after ourreview was accepted for publication: Van Roey et al., Science Signaling2013, http://dx.doi.org/10.1126/scisignal.2003345 and Talavera et al., NatureBiotechnology 2013, http://dx.doi.org/10.1038/nbt.2540.
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