MINIREVIEW
Alternative splicing: good and bad effects of
translationally silent substitutions
M. Raponi and D. Baralle
Academic Unit of Genetic Medicine, Human Genetics Division, University of Southampton, Southampton General Hospital, UK
Introduction
Splicing is an important part of a post-transcriptional
mechanism where introns are removed and exons are
joined together, allowing the resulting mature mRNA
to be translated into a specific protein product. This
mechanism is supported by the spliceosome machine,
which recognizes the well-characterized splicing con-
sensus sequences at the exon–intron junctions (donor
and acceptor sites) and their proximities (branch
points). Other cis-acting elements involved in the deter-
mination of the splicing outcome are recognized by
trans-acting factors that can either act as splicing
silencers or enhancers.
Alteration of splicing may occur whenever cis varia-
tions alter the recognition of splicing regulatory
sequences [1,2]. This could result in altered isoform
proportions, activation of a control mechanism such as
nonsense-mediated decay, as well as the creation or
loss of splicing variants. As this process has a signifi-
cant impact on protein abundance and ⁄ or functional-
ity, it follows that sequence variants in translationally
silent exonic positions that modify splicing are crucial
in genetic diagnosis and their role as a possible cause
of disease cannot be ignored. Equally important is the
role that these silent sequences may have in evolution.
For example, many algorithms used to calculate evolu-

may occur as a consequence of translationally silent mutations leading to
the expression of novel splicing isoforms and ⁄ or loss of an existing one.
This phenomenon can either generate new substrates for evolution or cause
genetic disease when aberrant isoforms altering the essential protein func-
tion are produced. In this review we briefly describe the current under-
standing in the field and discuss emerging directions in the study of the
splicing mechanism by integrating disease-causing splicing mutations and
evolutionary changes.
Abbreviations
K
a
, ratio of nonsynonymous substitutions; K
s
, ratio of synonymous substitutions.
836 FEBS Journal 277 (2010) 836–840 ª 2010 The Authors Journal compilation ª 2010 FEBS
changes in the alternative splicing (AS) isoforms (that
become substrates of both positive and negative
natural selection).
Two main categories of translationally silent varia-
tions can alter splicing: (a) intronic variations – changes
outside the coding exonic sequence; (b) synonymous
changes – variations that alter the exonic sequence, but
not the codon information, for an amino acid.
Translationally silent variations that
affect splicing and disease
Clinical studies identifying aberrant splicing mutations
are of great importance for genetic counselling, as a
good proportion of unclassified variants are often
found to be the cause of inappropriate RNA process-
ing (recently reviewed by Baralle et al. [3]). Such vari-

disruption of exonic ⁄ intronic splicing enhancer ⁄ silen-
cer sequences or creation of exonic ⁄ intronic splicing
silencer ⁄ enhancer sequences;
l
alteration of RNA secondary structure;
l
creation or disruption of splice sites;
In addition to the clinical importance of discovering
the aberrant effect of such mutations, they also repre-
sent an essential clue and wealthy resource for the
study of novel splicing regulatory mechanisms. There
is substantial precedence for identifying novel splicing
regulatory sequences and splicing factors by molecular
analysis of splicing aberrations caused by disease-caus-
ing mutations.
A good example of this was the mechanistic study
of a deep intronic GTAA deletion in the ATM gene
that permitted the identification of a novel intronic
splicing processing element [6]. Further functional
studies have shown how U1 binding to such intronic
elements can inactivate the inclusion of aberrant exons
[7].
Studies of this kind provide significant insights into
the splicing regulation of many genes, but this
approach has been poorly undertaken with regards to
synonymous changes that affect splicing.
Apart from synonymous variations causing disease
by creating or affecting the canonical splice sites, most
of them still lack experimental approaches directed at
identifying the exact mechanism involved. In spite of

M. Raponi and D. Baralle Alternative splicing
FEBS Journal 277 (2010) 836–840 ª 2010 The Authors Journal compilation ª 2010 FEBS 837
variations are not neutral for evolution. Understanding
this has important consequences in the way routine
diagnostic testing is approached.
In addition, the concept of non-neutrality for synon-
ymous variations will force an adjustment of the tradi-
tional way of measuring sequence evolution based on
the K
a
⁄ K
s
ratio (where K
a
is the ratio of nonsynony-
mous substitutions and K
s
is the ratio of synonymous
substitutions). This method, where the metric is based
on the neutrality of K
s
, has now become relatively
inaccurate. Although this approach is still in use,
researchers are aware that the K
s
may not always be
neutral, but is potentially affected by at least the splic-
ing constraint. As a consequence, a new approach has
emerged where the detection of K
a

tain correct splicing of the gene.
In fact, although we acknowledge that codon bias is
a potential index of splicing constraint, it should not
be forgotten that other selective forces may act at
silent sites, such as translational accuracy, mRNA
binding and mRNA stability (for a review see [14]). In
addition, missense variations can also affect splicing.
Therefore, a low K
a
may not only represent negative
selection at amino acid substitution, but also splicing
constraint. Therefore, the detection of both lower K
a
and K
s
in one region is probably an index of splicing
constraint rather than the detection of K
a
⁄ K
s
peaks,
which may be due to a high K
a
ratio and not to selec-
tive constraint at silent sites.
Notwithstanding the controversy surrounding the
measurement of purifying selection at silent sites, the
fact that synonymous substitutions are under selective
constraint because they have to ensure splicing effi-
ciency has already been experimentally demonstrated

The importance of clinical studies is not simply to
obtain important knowledge that a mutation has
caused a splicing defect, but also to provide a clue for
subsequent splicing functional studies, therapeutic
approaches and further elucidation of this complex
and interesting system. Evolutionary studies represent
another important field for the investigation of the ele-
ments involved in splicing regulation and the integra-
tion of all these approaches will give us the best
chance of finally understanding the splicing mechanism
itself.
An example from our own laboratory is the NF1
splicing mutation c.293–279A>G. This mutation was
Alternative splicing M. Raponi and D. Baralle
838 FEBS Journal 277 (2010) 836–840 ª 2010 The Authors Journal compilation ª 2010 FEBS
found to activate a pseudoexon and subsequent experi-
ments showed a novel mechanism by which the levels
of polypyrimidine tract binding proteins limit the
damaging pseudoexon inclusion [17]. The discovery of
such repression is of great relevance for further gene
therapy applications rescuing the patient’s wild-type
phenotype. This dependency on trans-acting factor
expression levels may also represent an important
observation with regards to explaining the variable
characteristics of disease, such as why particular
organs are affected by a mutation, age of onset,
individual susceptibilities, etc.
In addition, this discovery also brings insight into
the speculation that evolutionary changes may protect
against aberrant splicing due to a mutation as well as

fraction of transcripts where 67 nucleotides of intron
30 are retained [18]. Such tolerated splicing variants
can evolve freely in the pseudointronic sequence and
thus acquire a new function.
In conclusion, we need to reassess our view of nucle-
otide variations that were previously considered neu-
tral, particularly with regards to their effect on splicing.
A variety of tools are available to us for this purpose
and further investigation of these sequence variants will
not only further our understanding of the splicing
mechanism and improve clinical diagnostic testing, but
is also important for understanding gene evolution.
References
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