Two main categories of translationally silent varia-tions can alter splicing: a intronic variavaria-tions – changes outside the coding exonic sequence; b synonymous changes – variations
Trang 1Alternative 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-tionary distances are normalized against the transla-tionally ‘silent’ sequence variants, which until recently were considered evolutionarily neutral We now know that many so-called neutral substitutions are instead causative, as they produce the skipping of the exon or
Keywords
minigene; NF1; pre-mRNA; silent; splicing;
translation
Correspondence
D Baralle, Academic Unit of Genetic
Medicine, Human Genetics Division,
University of Southampton, Duthie Building
(Mailpoint 808), Southampton General
Hospital, Tremona Road, Southampton
SO16 6YD, UK
Fax: +44 2380794346
Tel: +44 2380796162
E-mail: D.Baralle@soton.ac.uk
(Received 26 August 2009, revised 4
November 2009, accepted 17 November
2009)
doi:10.1111/j.1742-4658.2009.07519.x
Nucleotide variations that do not alter the protein-coding sequence have been routinely considered as neutral In light of the developments we have seen over the last decade or so in the RNA processing and translational field, it would be proper when assessing these variants to ask if this change
is neutral, good or bad This question has been recently partly addressed
by genome-wide in silico analysis but significantly fewer cases by laboratory experimental examples Of particular relevance is the effect these mutations have on the pre-mRNA splicing pattern In fact, alterations in this process 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
Ka, ratio of nonsynonymous substitutions; Ks, ratio of synonymous substitutions.
Trang 2changes 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 variavaria-tions – 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-ants affecting splicing can be classified as pathogenic
mutations or genetic variations causing predisposition
to disease The first category usually has a devastating
effect on splicing, with a substantial loss of original
protein function or even acquisition of an antagonistic
function An explanation for the second category relies
on the fact that a weakly tolerated effect on splicing
can be enhanced by additional phenomena such as
affected expression of trans-acting factors that regulate
splicing [4]
Both intronic and synonymous nucleotide
substitu-tions can sufficiently alter splicing and cause genetic
dis-ease A list of silent mutations associated with altered
splicing was reported by Cartegni et al [5] However,
the number of examples reported to date in the
litera-ture is not as large as expected and this may be because
synonymous variations have previously always been
considered neutral and because of an existing bias to
search for alterations in the protein functional⁄
struc-tural properties Likewise, deep intronic variations that
do not affect the canonical splice sites have rarely been
taken into consideration or even reported owing to
technical difficulties in sequencing entire genes (also
until recently an expensive task) and in the
underappre-ciated belief that sequence variations far away may
affect far away splicing signals With increased
aware-ness we predict that this will change in the future
There are a number of different ways that such
variations may affect splicing, including:
l 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 esrepre-sential 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 the well-known lack of reliability of in silico approaches, many of the synonymous changes causing aberrant splicing are thought to alter exonic silencer⁄ enhancer sequences only on this basis [8,9] For instance,
a silent mutation in exon 7 of the POMGNT1 gene, in a patient with congenital muscular dystrophy, was shown
to promote skipping of this exon Here an extensive
in silicoanalysis predicted the creation of various splic-ing regulatory sequences, includsplic-ing an exon splice silen-cer, as well as a change in secondary structure [8] Another more characterized example comes from an
in silico analysis of two PDHA1 exon 5 silent variants Each variant determines exon 5 skipping and were pre-dicted to disrupt the splice enhancer SRp55 motif Using a minigene system, the inefficient exon 5 inclu-sion was corrected by strengthening the intron 5 donor site, suggesting that the putative SRp55 motif compen-sates for the weak donor site [9]
Translationally silent variations that affect splicing and evolution
With increasing understanding of the importance of cis regulatory sequences located either in the introns or in the coding sequence for the splicing process, the scien-tific world has become aware that there is a selective constraint for evolution, not only against sequence variations that alter the protein information, but also against variations that are harmful for the splicing pro-cess As the last category includes even supposedly silent changes, which do not alter the amino acid code, it follows that both intronic and synonymous
Trang 3variations 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 Ka⁄ Ks ratio (where Ka is the ratio of
nonsynony-mous substitutions and Ks is the ratio of synonymous
substitutions) This method, where the metric is based
on the neutrality of Ks, has now become relatively
inaccurate Although this approach is still in use,
researchers are aware that the Ks 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 Ka⁄ Ks ratio peaks in
genes, using a sliding window analysis, is assumed to
be an index of selective constraint acting on silent sites
An example of this type of approach is a fascinating
conservation analysis comparing BRCA1 orthologues
where the aligned coding sequences were used for two
independent sliding window analyses (mouse–rat and
human–dog) [10] This analysis showed a strong
puri-fying selection at silent sites in a critical region of this
gene [10], spanning the 3¢ end of exon 10 and the 5¢
end of exon 11 Purifying selection is the force that
drives negative selection to eliminate deleterious
muta-tions that would otherwise alter protein function The
possibility that this biased synonymous codon usage
reflects the necessity to maintain regulatory sequences
associated with splicing regulation was subsequently
suggested by the identification of two putative exon
splicing enhancers within the critical region [11]
However, this type of approach contains several
pit-falls, and it is important to acknowledge the existence
of recent bioinformatic studies showing that the
synon-ymous substitution rate reduction observed with the
sliding window analysis may often be artefactual
[12,13] As a result, there is a strong recommendation
that all these studies should be complemented by
fur-ther experimental support to demonstrate purifying
selection at silent sites in the gene of interest and to
demonstrate that such constraint is necessary to
main-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 Ka may not only represent negative
selection at amino acid substitution, but also splicing
constraint Therefore, the detection of both lower Ka
and Ks in one region is probably an index of splicing
constraint rather than the detection of Ka⁄ Ks peaks, which may be due to a high Ka 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 for the CFTR gene [15] In addition to reporting that
30% of the synonymous substitutions in human CFTR exon 12 significantly reduce its inclusion, this study has also brought new evidence that protein func-tion optimizafunc-tion can be constrained in exchange for the maintenance of proper splicing efficiency These results were confirmed by an additional evolutionary study that used CFTR exon 12 as a model and showed suboptimal composition at silent sites for splicing effi-ciency in the human exon and proposed a way by which exon loss may represent a substrate for evolu-tion when a combinaevolu-tion of synonymous changes induce partial exon skipping [16]
From an evolutionary point of view, however, the most frequently described substrate for natural selec-tion of new splicing variants is exon gain, although, as for exon loss, the precondition that allows a new splic-ing variant to evolve freely is the maintenance of origi-nal protein function Therefore, nucleotide variations that preserve the coding capacity (such as synonymous
or intronic substitutions), but also induce the inclusion
of a new exon in only a minor fraction of the mature transcript represent the best candidates in the creation
of new splicing substrates for evolution In this way, the newly generated alternative splicing exon has a bet-ter chance of being tolerated by the cell metabolism and is then free to evolve
Integrating evolutionary and splicing disease-related mechanistic studies –
an example
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
Trang 4found 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
predispose to disease Indeed, the same variation is
nor-mally present in the canine gene sequence where no
splicing alteration is observed As shown in Fig 1, we
demonstrated the compensatory relevance of some
nu-cleotides that differentiate the dog sequence from
human Canine nucleotide substitution in the human
minigene for splicing assay harbouring the c.293–
279A>G mutation was enough to mimic the normal
pseudoexon exclusion observed in dog and in human
normal phenotypes These data make it clear that
com-pensatory changes in dog protect against additional
variations that would produce intron exonization
Con-versely, in the human there is a predisposition to
muta-tions causing pseudoexon inclusion in NF1 intron 30,
which is only partially counteracted by the presence of
polypyrimidine tract binding protein binding sites
However, it would be wrong to conclude that
evolu-tionary changes happening in human introns should be
considered ‘bad’ because of a predisposition to
aber-rant splicing, as this may not represent the whole story
In fact, from an evolutionary point of view, the
procliv-ity of human intron 30 to exonize may be ‘good’ if
looked at as the ability to produce a new substrate for
evolution, as previously suggested [18] Indeed, the loss
of the intronic A>G variation from dog versus human, which creates a functional acceptor splice site only in combination with the human cryptic donor site 171 nu-cleotides downstream, has probably allowed the crea-tion of the latter Overall, this donor site is probably alternatively spliced in humans to produce a minor 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
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Fig 1 Human–dog inactivating substitutions completely repress pseudoexon inclusion The pseudoexon sequence in uppercase is compared with the dog (Canis familiaris) sequence Asterisks indicate nucleotide matches and dashes indicate sequence gaps The )279 a>g mutation and the nucleotide substitutions of human versus dog pseudoexon sequence are shown (t1 = G>T; t2 = A>T; g1 = A>G; g2 = T>G) Transfection in Hela cells of hybrid minigenes carrying both single and combinations of substitutions always show pseudoexon exclusion (data not shown).
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