alternative splicing of intrinsically disordered regions ... during evolution, development and...

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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 Babu1
Alternatively 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


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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


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


http://www.rcsb.org/

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


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


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).



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)


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


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



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/


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].



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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alternative splicing of intrinsically disordered regions ... during evolution, development and...

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