Quantitative analysis of wobble splicing indicates
that it is not tissue specific☆
Kuo-Wang Tsaia,b, Wen-chang Linb,c,⁎
aGraduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
bInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China
cInstitute of Bioinformatics, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
Received 27 April 2006; accepted 2 July 2006
Available online 22 August 2006
Alternative splicing is an important mechanism mediating the function of genes in multicellular organisms. Recently, we discovered a new
splicing-junction wobble mechanism that generates subtle alterations in mRNA by randomly selecting tandem 5′ and 3′ splicing-junction sites.
Here we developed a sensitive approach to identify such splicing-junction wobble isoforms using polymerase chain reaction amplification with
fluorescence-labeled primers encompassing the wobble-splicing boundary and capillary electrophoresis. Using the ING4 wobble isoforms as an
example, we demonstrated that capillary electrophoresis can precisely separate DNA fragments with a small difference in size (<3 nt) and can be
used to quantify the expression ratio, which thus measures the distribution of each splicing-junction wobble isoform in tissues. Based on our
analyses of several genes, the relative ratio of each wobble-splicing isoform tends to be constant among various tissues. The occasional observed
tissue heterogeneity of wobble-splicing transcripts can be generated only by genomic single-nucleotide polymorphisms around the splicing
© 2006 Elsevier Inc. All rights reserved.
Keywords: Alternative splicing; Wobble splicing; Splicing junction; ING4; Capillary electrophoresis; SNP
Alternative splicing is a main source of transcriptome and
proteome diversity . Over the past few years, the analysis of
alternative splicing using bioinformatics has emerged as an
important new topic [2–6]. Recently, Hiller et al.  and
Tadokoro et al.  reported interesting results showing that
splicing acceptors with the genomic NAGNAG motif can cause
NAG insertion–deletion (InDel) in transcripts and may play
surprisingly complex roles in switching protein conformation
and function. We recently used a bioinformatics approach to
discover putative single-amino-acid InDel variants by searching
and analyzing the human expression sequence tag (EST)
database . The NAGNAG-based tandem repeat was the
major splicing-junction wobble site observed in our previous
study, but our results also showed that the splice donor
(GTNGT) and non-NAGNAG splice acceptor sites can also
be used to generate such InDel variants in addition to bona fide
genomic DNA InDel events.
As an example in our finding, inhibitor of growth 4 (ING4), a
well-known tumor suppressor gene, contains a wobble-splicing
region. ING4 contains GC(N)7GT and NAGNAG motifs at the
exon 4-5 boundary, which could cause canonical (GT-AG) and
noncanonical (GC-GT) splicing site wobbling selection (Fig.
1A) . Using database searches, we found that the GC(N)7GT
and NAGNAG motifs were conserved in human and mouse
ING4. Additional searches of GenBank revealed that human
ING4 exists in two transcripts (BC007781:GKKKG and
BC095434:GKKS), as does mouse ING4. In addition, Kim et
al.  found a common deletion in ING4 transcripts from
various human cancer cell lines, which consists of 12 nt (at the
exon 4-5 boundary), which eliminates a sequence of four amino
acids (KGKK). It is not clear whether the deletion represents a
polymorphism or is the result of alternative splicing. These
subtle changes in splicing site selection may increase ING4
protein diversity in a wide range of species.
Genomics 88 (2006) 855–864
☆This paper contains supplementary data that can be found at http://
⁎Corresponding author. Fax: + 886 2 27827654.
E-mail address: email@example.com (W. Lin).
0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
Alternative splicing is often found to be tissue specific or
developmental stage specific. Recent reports have also shown
that several genes are alternatively spliced at NAGNAG
acceptors and that this event might be tissue specific [4,7].
These studies could not, however, fully ascertain the tissue
specificity because of limitations of the RNA sources and
analysis techniques related to the low expression level of
wobble-spliced transcripts . PCR-restriction fragment length
polymorphism (RFLP) and reverse transcription (RT)-PCR
polyacrylamide gel electrophoresis methods are the two
methods that have previously been used to verify and quantify
the subtle variants resulting from wobble splicing [7,8].
Unfortunately, these methods are time consuming, unable to
quantify the percentages of subtle splicing variants, and difficult
to standardize across animal species.
Because of the minor effects on mRNA and protein structure
that are caused by wobble splicing, this phenomenon is
frequently overlooked or underestimated in laboratory experi-
ments. Herein we introduced a modified capillary electropho-
resis (CE)–based approach for the identification and
quantification of the four alternative splicing-junction wobble
isoforms of ING4. Furthermore, we have applied this powerful
technique to other genes and observed that the greater part of
wobble splicing at tandem motifs is not controlled in a tissue-
Wobble-splicing isoforms in ING4
The ING4 genomic sequence contains the GC(N)7GT and
NAGNAG motifs, which may produce alternative 5′ and 3′
splicing sites at the exon 4-5 boundary. If all possible
combinations of the two individual alternative splicing events
are taken into account, four different splicing variants of ING4
would be expected (Fig. 1A). By searching the EST database,
we identified four different subtle splicing-junction wobble
isoforms of ING4 located at the exon 4-5 boundary. In the
human EST database, we scanned 86 ING4 ESTs and identified
the isoforms GKKKG (29 ESTs, 33.7%), GKKS (41 ESTs,
47.7%), EG (3 ESTs, 3.5%), and G (13 ESTs, 15.1%). In the
Fig. 1. Verification of ING4 wobble-splicing using the capillary electrophoresis approach. (A) ING4 alternatively spliced mRNA variants, ING4GKKKG, ING4GKKS,
ING4EG, and ING4G. The four different isoforms of ING4 are caused by alternative splicing at the 5′ tandem donor [GT(N)7GC] and 3′ tandem acceptor (NAGNAG)
at the exon 4-5 boundary. (B) Schematic diagram showing the PCR primer position for amplification of the ING4 wobble-splicing variants. The single-headed arrows
show the primer position, and the double-headed arrow shows the overall PCR product size using this particular primer set. The PCR products created for the different
wobble-splicing isoforms had different sizes. The reverse primer was labeled with fluorescent dye (FAM) to label the PCR product. (C) Schematic illustration of the
separation and detection of the four ING4 wobble-splicing isoforms by capillary electrophoresis (blue peaks). The reference peaks of the GS350 ROX internal size
standard (Applied Biosystems, Foster City, CA) are indicated in red, with the molecular weights indicated below the peak.
856K.-W. Tsai, W. Lin / Genomics 88 (2006) 855–864
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