Full text of DOI:10.1093/nar/29.19.4006

4006–4013 Nucleic Acids Research, 2001, Vol. 29, No. 19
© 2001 Oxford University Press
A computational scan for U12-dependent introns in the
human genome sequence
Aaron Levine and Richard Durbin*
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
Received June 8, 2001; Revised and Accepted August 1, 2001
ABSTRACT
GT-AG terminal dinucleotides as well as a few with other
termini (6–8). Additionally, a small number of U2-dependent
introns with U12-like AT-AC terminal dinucleotides have
been identified, confirming that analysis of the entire splice
site signal and not just the terminal dinucleotides is required
for accurate classification (9).
Many genes contain both U2- and U12-dependent introns
but little is known about how the two spliceosomes cooperate
to identify and splice the correct introns in vivo. Distinct differ-
ences are observed, however, between the splice site signals
associated with the two types of introns. U12-dependent
introns exhibit strongly conserved and informative donor and
branch signals, whereas U2-dependent introns exhibit only
moderately informative signals at the donor and acceptor sites
and a highly degenerate branch site signal. Additionally the
polypyrimidine tract seen between the branch site and acceptor
site of U2-dependent introns is lacking in U12-dependent
introns (1).
The evolutionary history of these two classes of introns and
their respective spliceosomes remains unclear. Burge et al. (7)
have reported AT-AC U12-dependent introns converting to
GT-AG U12-dependent introns and U12-dependent introns
converting to U2-dependent introns and concluded that
U12-dependent introns tend to convert to U2-dependent over
evolutionary time. They also reported a biased distribution of
U12-dependent introns within a variety of genomes, a result
they found suggestive of a fission–fusion model of spliceo-
some evolution in which the U2 and U12 systems diverged in
separate lineages and were later united through a merging of
genetic material in a progenitor of higher eukaryotes (7).
Recent results have found a strikingly high degree of overlap
between the proteins and non-coding RNAs involved in U2-
and U12-dependent splicing. In addition to the U5 snRNA
(10), all eight snRNP Sm proteins (11), the four proteins that
constitute the splicing factor SF3b (11), and the splicing-
associated protein Prp8 have been found in both the U2 and
U12 spliceosomes (12). Recent evidence has indicated that
splicing-associated SR proteins, long known to function in the
major spliceosome, play functional roles in U12-dependent
splicing as well (13). Extensive similarity in secondary struc-
tures and interactions between the set of non-coding RNAs
U11, U12, U4atac and U6atac involved in the U12 spliceo-
some and the set U1, U2, U4 and U6 involved in the U2
spliceosome argue for homology of the two systems as well (7)
as do recent results that have found the stem–loop structures of
U6 and U6atac to be functionally analogous (14). Although the
evolutionary implications of this high degree of overlap are not
U12-dependent introns are found in small numbers in
most eukaryotic genomes, but their scarcity makes
accurate characterisation of their properties chal-
lenging. A computational search for U12-dependent
introns was performed using the draft version of the
human genome sequence. Human expressed
sequences confirmed 404 U12-dependent introns
within the human genome, a 6-fold increase over the
total number of non-redundant U12-dependent
introns previously identified in all genomes.
Although most of these introns had AT-AC or GT-AG
terminal dinucleotides, small numbers of introns
with a surprising diversity of termini were found,
suggesting that many of the non-canonical introns
found in the human genome may be variants of
U12-dependent introns and, thus, spliced by the
minor spliceosome. Comparisons with U2-dependent
introns revealed that the U12-dependent intron
set lacks the ‘short intron’ peak characteristic
of U2-dependent
introns.
Analysis
of
this
U12-dependent intron set confirmed reports of a
biased distribution of U12-dependent introns in the
genome and allowed the identification of several
alternative splicing events as well as a surprising
number of apparent splicing errors. This new larger
reference set of U12-dependent introns will serve as
a resource for future studies of both the properties
and evolution of the U12 spliceosome.
INTRODUCTION
Two distinct types of pre-mRNA introns, termed U2- and
U12-dependent based on the spliceosome complexes that
excise them during RNA processing, are found in most higher
organisms (reviewed in 1). While the 99.9% of introns spliced
by the major (U2-dependent) spliceosome have been exten-
sively characterised (2,3), less is known regarding the
remaining 0.1% of introns, which fall into the U12-dependent
class. This minor class of introns was originally identified due
to its unusual conserved donor and branch signals and highly
atypical AT-AC terminal dinucleotides (4,5). More recently,
analyses have found that AT-AC termini are not strictly
required and identified many U12-dependent introns with
*To whom correspondence should be addressed. Tel: +44 1223 834244; Fax: +44 1223 494919; Email: rd@sanger.ac.uk

Nucleic Acids Research, 2001, Vol. 29, No. 19 4007
entirely clear, these findings may indicate that the U12 spliceo-
some evolved in the presence of the U2 spliceosome rather
than in a different lineage as the fission–fusion model suggests
(11).
Table 1. Diverse terminal dinucleotides on U12-dependent introns in the
human genome
Intron termini Reported in
Total found
Putative
Total
Burge et al. (7)
splicing errors confirmed
U12-dependent introns have been identified previously
through homology searches and by analysing annotated intron
junctions (7). The analysis presented here represents the first
large-scale search for U12-dependent introns in the recently
completed human genome sequence. A greater than expected
diversity in the terminal dinucleotides of U12-dependent
introns was observed, giving further evidence to the idea that
flexibility in these positions has played an important role in
intron evolution. This analysis generated a new reference set of
human U12-dependent introns 8-fold larger than the previously
available set and allows a more extensive characterisation of
these introns to be carried out.
GT-AG
AT-AC
AT-AG
GT-AT
AT-AT
GT-GG
AT-AA
GT-AA
GT-CA
GC-AG
Totals
34
12
1
279
109
8
4
1
275
108
7
1
0
5
1
4
0
4
0
4
0
7
4
3
1
5
3
2
0
1
0
1
0
1
1
0
1
0
0
0
49
419
15
404
MATERIALS AND METHODS
Human U12-dependent introns were identified using a
two-step procedure
The total number of U12-dependent introns previously reported in Burge et al.
(7), the original total found in this analysis, the total discarded as likely splicing
errors and the final confirmed total are shown for a variety of possible intron
termini. Genomic scans were also performed for introns with GT-AC, GC-AC,
GG-AC, GG-AG, AT-GG and AT-CA termini. No U12-dependent introns
were identified in these scans. The AT-AC and GT-AG scans considered
target introns with donor site scores greater than nine bits, branch site scores
greater than six bits and lengths of <20 kb. All other scans examined introns
of up to 2 kb in length. The GT-AC and AT-AG scans used 11 and nine bits as
donor and branch scores thresholds, the AT–AA scan used 10 and eight bits
and all remaining scans used 10 and seven bits. All scans used a branch site to
acceptor site range of 8–21 bp.
First, potential donor and branch site signals were identified
based on statistical pattern recognition techniques. Low
threshold values that detected almost all known sites while
accepting a large number of false positives were used. From
these signals, potential introns (donor/acceptor pairs) were
generated and expressed sequence evidence was used to iden-
tify a subset of these potential introns as valid. All genomic
scans used the 9 January 2001 assembly of the 7 October 2000
freeze of the human genome draft sequence (2; available from
http://genome.cse.ucsc.edu/).
Candidate U12-dependent intron donor and branch sites
were identified using a standard weight matrix approach (15).
The weight matrix models were trained using a previously
described non-redundant set of 48 U12-dependent introns from
a variety of species (6). Simple pseudocounts based on
genomic nucleotide frequencies (the null model) were added
during the training process to avoid overfitting the model to the
training set. Any sequences whose log-odds scores from the
donor signal weight matrix exceeded an empirically derived bit
threshold were considered potential U12-dependent intron
donor sites. Potential U12-dependent acceptor signals were
identified by considering all high-scoring branch signals (again
using an empirically derived threshold) and including only
those that had a putative acceptor site (an AC dinucleotide, for
instance) within a certain distance range from the putative
branch site. The traditional consensus branch site for
U12-dependent introns is TTCCTTAA, although our search
pattern extended slightly beyond this consensus and none of
the bases were strictly required in our analysis. All potential
donors and acceptors that met the above criteria and were
within a certain distance of each other were considered to
define a potential U12-dependent intron. For each of these
cases, 64 bp of potential exon sequence, 32 bp from before and
32 bp from after the hypothetical intron were extracted and
saved for later analysis.
intron size (20 kb). The first four of these were selected to be
as inclusive as possible (based on the training data) while still
minimising time required for computation, while the final
parameter, maximum intron size, had to be limited to relatively
small values to render the analysis computationally tractable.
The analysis did, therefore, overlook some longer U12-
dependent introns (see Discussion). After confirmation of
introns, the distributions of donor scores, branch scores and the
branch to acceptor distance were plotted and showed approxi-
mately normal distributions with the thresholds well separated
from the peaks (see Fig. 2 and data not shown), suggesting that
our empirical thresholds did not eliminate a large number of
valid results. Parameter values for the GT-AG and AT-AC
scans are provided above; parameter values for all scans are
provided in the legend to Table 1.
Expressed sequence data were used to confirm a small
portion of the large set of potential U12-dependent introns as
true introns. For this purpose a specialised human expressed
sequence database was developed which contained 54 484
human RNA sequences from EMBL release 65 (16) and 3 268
161 human ESTs from dbEST downloaded from the NCBI on
28 February 2001 (17; available from ftp://ncbi.nlm.nih.gov/
genbank/).
High-speed SSAHA similarity searches were performed
looking for matches between each potential U12-dependent
intron and a repeat-masked version of the database described
above (18). Repeat masking was performed using DUST
The analysis described above involved five parameters: a
donor site score threshold (nine bits), a branch site score
threshold (six bits), both a minimum and a maximum branch
site to acceptor site distance (8 bp, 21 bp) and a maximum

4008 Nucleic Acids Research, 2001, Vol. 29, No. 19
(R.L.Tatusov and D.J.Lipman, unpublished). SSAHA (version
1.1) was used with the following options: wordlength, 13;
minprint, 39; maxstore, 50000; reportmode, replaceC. The
results of this search were parsed to include only those
expressed sequence matches that extended at least 15 bp on
both sides of the hypothetical splice junction. Two potential
introns were considered duplicate if they showed identical
sequences along the full 64 bp of potential exon regions.
Although such a situation could potentially result from gene
duplications and represent a valid intron, redundancy in the
draft sequence assembly presents an equally plausible explana-
tion. Accordingly only one copy of each potentially duplicate
intron was saved for further analysis. Introns supported by a
variety of SSAHA matches extending from at least position 3
to position 61 were considered verified at this point. As
SSAHA functions in a phased manner and does not necessarily
report the full length of the sequence match, introns which
showed support but did not meet this stringent SSAHA criterion
were analysed using BLAST (19; version 2.0.6, installed
locally). Introns supported by a perfect BLAST match over all
64 bp were considered as verified. The remaining set of candi-
date introns, which showed some support but met neither the
SSAHA nor the BLAST criteria were examined and classified
manually.
Scans were performed for the standard U12-dependent
introns with AT-AC and GT-AG terminal dinucleotides as
well as a variety of non-standard introns (Table 1). Non-
standard donor signals were identified using modified training
sets, which had, for instance, each GT dinucleotide at the
donor position replaced with a GC dinucleotide. Non-standard
acceptors were identified by using the original branch site
training set but scanning the downstream region after high-
scoring branch sites for the non-standard dinucleotide of
interest.
using the stringent criteria described above. Five out of these
388 were classified as likely splicing errors and removed
from further analysis. The 383 AT-AC and GT-AG human
U12-dependent introns reported here represent an increase of
337 (>8-fold) over the introns reported in the only similar
study (7).
In total, scans for U12-dependent introns with 16 different
combinations of terminal dinucleotides were performed (Table 1).
A total of 419 introns, including the 388 AT-AC and GT-AG
introns discussed above, met the confirmation criteria. Of the
additional 31 introns, 10 were classified as likely splicing
errors, leaving a total of 21 non-AT-AC or GT-AG human
U12-dependent introns, distributed among six classes,
including the previously documented AT-AG and AT-AA (7)
as well as several previously undocumented classes. Examina-
tion of the donor and acceptor signals of the atypical
U12-dependent introns reveals almost perfect conservation of
both the donor and branch sites with the U12-dependent intron
consensus sequences. Detailed information, including intron/
exon junction sequences, for all 404 confirmed introns is
available as Supplementary Material in the online version of
the paper or from http://www.sanger.ac.uk/Users/rd/U12/.
Despite searches for introns starting with GC or GG, all
confirmed introns showed standard AT or GT dinucleotides at
the donor position, suggesting that these bases may be almost
universally required for successful splicing. One GC-AG
U12-dependent intron, which was missed during our analysis
due to its atypical and low-scoring donor site, has been
reported previously indicating that an AT or GT dinucleotide is
not an absolute requirement (7). In contrast, a variety of
terminal dinucleotides (including AG, AC, AT, AA and GG)
were observed at the acceptor position. The diversity of
terminal dinucleotides observed at the acceptor site of human
U12-dependent introns confirmed recent experimental work,
which indicated that a variety of dinucleotides can serve as
functional U12-dependent acceptor sites in vitro (21). This
flexibility fits well with the idea that the branch site serves as
the primary recognition point for the 3′ end of U12-dependent
introns and suggests that the mechanism of 3′ site identifica-
tion may be only loosely constrained.
282 confirmed GT donor sites were also scored as
U2-dependent donor sites, using a custom U2 splice predictor
based on first order weight matrices (A.Levine, unpublished;
22). The vast majority of these sites scored poorly as U2 sites.
Only seven out of 282 (2.5%) received a log-odds score greater
than five bits and even these scores were generally well below
the mean score (mean 8.66, SD 2.31) for a set of 3620 true sites
scored with the U2 model.
Estimating the frequency of U12-dependent introns within
the genome is a difficult problem and due to the lack of
comparable data for U2-dependent introns our results do not
lead to an easy solution. However, comparing the small sample
of 11 U12-dependent introns we identified on chromosome 22
with the 3199 U2-dependent introns identified in a similar
search for U2-dependent introns on chromosome 22 suggests
that as many as 0.34% of human introns are spliced by the U12
spliceosome. This number is larger than earlier estimates that
suggested roughly 0.15% of human introns were likely to be
U12-dependent (7), but, due to the small sample size, must be
taken as only a rough estimate.
Non-standard splice junctions were checked for possible
ambiguities in the form of cases where a single expressed
sequence could support a variety of splice junctions, as previ-
ously described (20). No such cases were found.
Distributions of U12-dependent introns in the genome were
modelled using binomial distributions as previously described
(7).
RESULTS
Characteristics of human U12-dependent introns
Scans of recent human genome draft sequence were performed
to identify both typical AT-AC and GT-AG U12-dependent
introns and atypical U12-dependent introns with a variety of
other splice junctions (Table 1). The searches for AT-AC and
GT-AG introns examined all candidate introns up to 20 kb in
length while the other searches only examined potential introns
of up to 2 kb in length. Accordingly atypical introns are likely
to be somewhat under-represented in our results. Unlike the
only previous large-scale U12-dependent intron search, these
scans analysed unannotated genome sequence data and were
neither biased nor aided by previous annotation (7).
The search for AT-AC and GT-AG introns examined
∼20 million candidate introns found by pairing high-scoring
U12-dependent donor and branch site splice signals. Of these
candidates, 388 were confirmed by expressed sequence data

Nucleic Acids Research, 2001, Vol. 29, No. 19 4009
Table 2. Phase of U2- and U12-dependent introns
U2-dependent
introns
U12-dependent
introns
Number
5263
Percentage
47.4
Number
59
Percentage
20.8
Phase 0
Phase 1
Phase 2
Total
3372
30.3
118
41.5
2482
22.3
107
37.7
11 117
100
284
100
The number and percentage of U2- and U12-dependent introns that fall into
the three possible phase classes are shown. Introns are phase 0 when the intron
occurs between codons, phase 1 when the intron is found 1 base into the
codon and phase 2 when the intron is found 2 bases into the codon.
U2-dependent intron data is from Long et al. (23).
Figure 1. Length of U12- and U2-dependent introns. The length of 168 U12-
dependent introns and 11 402 RefSeq-confirmed U2-dependent introns <1 kb
in length are plotted. Grey bars represent the counts of U12-dependent introns
grouped into 50-bp wide bins, while the black line represents the frequency of
U2-dependent introns grouped into 10-bp wide bins.
U12-dependent introns than it is for U2-dependent introns and
verify suggestions (21) that AT-AC and GT-AG introns show
different distributions for this distance (chi-squared test,
P < 0.001). No functional relevance for this difference has
been identified.
Table 2 compares the phase of 284 of the U12-dependent
introns found in this study with 11 117 predominately
U2-dependent introns previously analysed (23). The two
distributions differ significantly (chi-squared test: P < 0.001)
with the most striking difference being the bias against phase 0
introns in the U12-dependent intron data, compared with the
bias toward phase 0 introns in the U2-dependent intron data.
These results generally agree with previously analysed intron
phase data from a smaller dataset (7).
We built frequency tables for the donor, branch and acceptor
site of both AT-AC and GT-AG U12-dependent introns from
our large set of these introns and provide these as Supplementary
Material. Although some slight differences are evident, the
consensus sequences for these sites largely agree with those
derived previously (1).
Figure 2. Branch site to acceptor site distance for U12-dependent introns. The
distance between the branch site and the acceptor site is plotted for 108 AT-AC
U12-dependent introns (black bars) and 275 GT-AG U12-dependent introns
(grey bars).
Analysis of the region between the branch site and the
acceptor site reveals a slight pyrimidine bias in this region.
Sixty-six percent of a sample of 2191 nt from between the
branch and acceptor consensus sequences were pyrimidines,
while only 54% of a control set of 3060 nt from upstream of the
branch site consensus sequence were pyrimidines. Although
extracting a comparable set of data for U2-dependent introns is
difficult, pyrimidines make up nearly 80% of the nucleotides in
the 9 bp upstream of the acceptor site consensus (CAG),
suggesting that the pyrimidine bias at U12-dependent introns is
not as strong as it is at U2-dependent introns.
Access to this large set of confirmed U12-dependent introns
allowed us to analyse several characteristics of this rare class
of introns. Figure 1 compares the length distribution of the 168
confirmed U12-dependent introns with 11 402 U2-dependent
introns from version 1.0 of Ensembl (2). U2-dependent introns
have a two-component distribution, with a peak at ∼90 bp
and an exponential-like component for longer lengths.
U12-dependent introns seem to be lacking the short component
of the U2-dependent intron length distribution. In contrast,
U12-dependent introns show a gradual peak between 200 and
250 bp, then a slow decay. The distributions are similar for
larger introns between 1 and 20 kb (U2: mean 4130 bp, SD
3720 bp; U12: mean 3600 bp, SD 3300 bp and data not shown),
showing that the exponential components are similar.
High error rates at the acceptor site in U12-dependent
splicing
A surprisingly high number of introns were identified which
met all confirmation criteria, yet seemed unlikely to represent
real introns. In general, these introns shared donor sites with
other confirmed introns yet differed slightly (1–6 bp) in
acceptor site positions. In most cases one member of these
pairs of introns had typical terminal dinucleotides and was
strongly supported by a large number of expressed sequences
while the second exhibited atypical dinucleotides and was
The distribution of the distance between the branch site and
the acceptor site for both AT-AC and GT-AG U12-dependent
introns is illustrated in Figure 2. These results confirm earlier
findings that this distance is much more sharply restricted in

4010 Nucleic Acids Research, 2001, Vol. 29, No. 19
Table 3. Putative 3′ splicing errors in U12-dependent splicing
Interestingly, three of these alternative splicing events
involved introns with different pairs of terminal dinucleotides.
For instance 14 expressed sequences supported an AT-AT
intron of length 620 bp in a hypothetical human protein
(DDBJ/EMBL/GenBank accession no. NM_024549) while
two expressed sequences support an AT-AC intron with the
same donor site but a different acceptor site 3344 bp down-
stream of the donor site.
These results suggest that, at a minimum, 13 out of 404 or
~3.2% of human U12-dependent introns show truncation/exten-
sion type alternative splicing events. A bias (11 out of 13)
towards alterations at the acceptor site was also observed,
although the numbers are too small to draw any strong conclu-
sions in this regard. A similar analysis of ∼3200 expressed-
sequence-confirmed U2-dependent introns (of length <20 kb)
on human chromosome 22 found truncation/extension alternative
splicing events to occur at ~14% of introns and only negligible
differences between the frequency of events involving donor
and acceptor sites (data not shown).
Confirmed intron
ID Termini
Putative splicing error
3′ Difference
Evidence
Termini
GT–GG
GT–CA
GT–AG
GT–GG
AT–AC
GT–AG
GT–GG
GT–AT
AT–AA
AT–AA
AT–AA
GT–GG
GT–AG
AT–AG
GT–AG
Evidence
2
GT-AG
GT-AG
GT-AG
GT-AT
AT-AC
GT-AG
GT-AG
GT-AG
AT-AC
AT-AC
AT-AC
GT-AG
GT-AG
AT-AC
GT-AG
8
94
7
1
1
1
1
1
1
7
1
1
1
1
1
1
1
1
–4
–1
–3
+3
+4
+2
+1
–2
+5
–3
+6
–3
–4
+2
+2
14
45
92
2
97
7
122
124
127
145
216
226
236
251
290
393
60
266
12
12
16
3
15
7
Non-random distribution of U12-dependent introns in the
genome
13
24
The distribution of U12-dependent introns within the human
genome has important implications for understanding the
evolutionary history of the major and minor spliceosomes.
Among the 404 U12-dependent introns identified in
this analysis, there were 16 cases where two or more
U12-dependent introns were confirmed by the same expressed
sequence, indicating that the two introns occurred within a
single gene (Table 5). One of these cases (Homo sapiens NHE-6,
DDBJ/EMBL/GenBank accession no. AF030409) had three
U12-dependent introns (one AT-AC, two GT-AG) supported
by a single expressed sequence.
If we assume that U12-dependent introns are randomly
distributed throughout the genome, the probability of identi-
fying 16 or more genes with multiple U12-dependent introns
among 388 genes with at least one U12-dependent intron is
P < 0.009. This strongly confirms earlier reports that suggested
U12-dependent introns were distributed non-randomly within
genomes (7). It is worth noting that the strict requirement for
multiple introns to be supported by a single expressed
sequence almost certainly leads to an underestimate of the true
number of genes with multiple U12-dependent introns and,
thus, an overestimate of the likelihood of this distribution
occurring by chance. This underestimation occurs due to the
short length of most ESTs and the correspondingly small
chance that a single EST would support multiple introns.
Furthermore, in this analysis duplicate U12-dependent introns,
which arose from gene duplications during evolution, are
counted as distinct introns. If each group of duplicate introns
was counted as a single intron, the likelihood of seeing this
distribution arising randomly would be reduced.
The total evidence (number of expressed sequences) supporting a confirmed
intron and an associated putative splicing error are shown. Each confirmed
intron and associated splicing error share the same donor position but differ by
a small number of bases (3′ difference) in the acceptor position. The ID column
corresponds to the ID field on the complete intron list provided as Supplementary
Material.
weakly supported. In many cases the second intron led to the
subsequent exon being out of frame and thus is unlikely to
represent a true alternatively spliced variant of the gene.
Fifteen introns exhibited these criteria and were classified as
likely splicing errors (Table 3). Although a few of these so-
called splicing errors may represent errors in EST sequencing,
most seem likely to represent mistakes made by the U12-
spliceosome.
In total, 21 ESTs were observed confirming likely splicing
errors and 5864 ESTs were observed confirming accepted
introns. These numbers suggest that splicing mistakes at the
3′ end of U12-dependent introns occur at a rate of approxi-
mately one error in every 280 splices. This value likely under-
estimates the true error rate in U12 acceptor site selection as
our analysis considered only a small subset of possible
terminal dinucleotides. Similar genomic scans with other pairs
of bases at the acceptor position could potentially uncover
even more evidence of errors during U12-dependent splicing.
Alternative splicing of U12 introns
The approach to intron identification used for these analyses
allowed us to identify alternative splicing situations in which
one splice site was used in two or more confirmed introns.
Among the 404 U12-dependent introns, 13 cases of such alter-
native splicing events were observed (Table 4). Eleven cases
were identified where the same donor signal was used with a
different acceptor signal and two cases were found in which
different donor signals were paired with the same acceptor site.
DISCUSSION
The analysis presented here greatly increases both the number
of U12-dependent introns identified and the diversity of these
introns. The observation that a significant number of
U12-dependent introns exhibit atypical terminal dinucleotides
suggests that a good number of the so-called non-canonical
introns identified in a variety of genomes (24) may represent

Nucleic Acids Research, 2001, Vol. 29, No. 19 4011
Table 4. Alternatively spliced U12-dependent introns
Gene
ID
Termini
GT-AG
GT-AG
AT-AC
AT-AC
GT-AG
GT-AG
GT-AG
Length
1145
1593
4501
4522
2403
3187
103
Evidence
Accession no.
X04217
Porphobilinogen deaminase (PBG-D)
3
26
2
4
343
342
R06263
Quinone oxidoreductase homolog-1
3
AA370151
AF029689
U96759
11
35
1
Von Hippel–Lindau binding protein (VBP-1) 132
133
BF667071
AJ251367
Calcium channel, alpha 2/delta subunit 2
(CACNA2D2)
247
2
248
105
106
304
303
158
157
287
288
67
GT-AT
GT-AG
GT-AG
GT-AG
GT-AG
AT-AC
AT-AT
GT-AG
GT-AG
GT-AG
GT-AG
AT-AC
AT-AC
GT-AG
GT-AG
GT-AG
GT-AG
GT-AG
AT-AG
97
2951
5038
13385
13423
3344
620
4
2
AF042972
AV725561
AI917412
BF373273
BE887649
AK024780
BE275895
AK001916
BE263460
T50022
Unknown
1
Unknown
1
2
Unknown
2
14
39
1
Unknown
1471
2747
605
Unknown
17
1
68
2677
8926
277
AL523899
AF077188
AL560997
AK000443
AK022732
L26318
Cullin 4a (CUL4A)
Unknown
257
258
386
387
367
368
106
107
21
1
12503
14540
1727
1301
5038
1984
2
4
JNK1 protein kinase
Unknown
2
3
L35004
1
AI917412
AI023856
5
Thirteen examples of alternatively spliced U12-dependent introns are shown. For each splicing variant, the ID matching the Supplementary intron
table, the intron terminal dinucleotides, the intron length, the total evidence supporting the intron and an accession number of a confirming
expressed sequence are presented.
variants of U12-dependent introns. Furthermore, due to the
different parameters used in the searches for typical and
atypical U12-dependent introns, the results presented here
most likely reflect an under-representation of atypical
U12-dependent introns. For instance, only 76% of AT-AC and
GT-AG U12-dependent introns have lengths <2 kb. If this ratio
holds for atypical U12-dependent introns as well, the 21 examples
reported here should increase to roughly 27. Furthermore,
scans for introns with pairs of terminal dinucleotides not
considered in this study may identify additional atypical
U12-dependent introns as well.
The 404 U12-dependent introns identified here represent a
lower bound on the genome’s full complement of these introns
for a variety of reasons. First, as noted previously, the arbitrary
limit of 20 kb as the maximum intron length for AT-AC and
GT-AG U12-dependent introns almost certainly excluded a
significant number of true introns from our analysis. For
comparison, ~5% of Ensembl U2-dependent introns confirmed
by RefSeq entries are >20 kb in length (2). In addition, the
threshold values used for donor and branch site scores, while
chosen to be inclusive, likely excluded a small number of valid
introns from the analysis.
Furthermore, the incomplete nature of the EST and mRNA
sets used to confirm introns means that some number of true
introns, which were identified as potential introns in the first
stage of this analysis, failed to meet the confirmation criteria
and were not included in the final counts. EST datasets in
particular are biased towards the 5′ and 3′ ends of genes and are
less likely to provide evidence for introns near the middle of
larger genes.
A large majority of the human U12-dependent introns
reported previously were identified in our large-scale genomic
analysis. However, a few appear to have been missed. In
addition to the GC-AG U12-dependent intron discussed above,
intron 5 of FHIT (human fragile histidine triad gene) and intron

4012 Nucleic Acids Research, 2001, Vol. 29, No. 19
16 of HPS (human Hermansky–Pudlak syndrome gene),
previously noted to be U12-dependent introns (7), were both
missed by our analysis. Careful examination of these particular
introns reveals that the FHIT intron was missed due to its
exceptionally long length while the HPS intron was missed to
due to its atypical and low-scoring donor and branch sites.
The large set of U12-dependent introns presented here
should prove helpful for future studies regarding the evolution
of the two-spliceosome system. In particular comparisons with
the nearly complete mouse genome should prove useful in
analysing the frequency of subtype switching between AT-AC
and GT-AG U12-dependent introns, as well as intron conver-
sion and loss.
Table 5. Genes with multiple U12-dependent introns
Gene
U12-dependent introns Accession no.
Smg GDS-associated
protein (SMAP)
GT-AG (84)
AT-AC (85)
U59919
Transcription elongation
factor TFIIS.h
AT-AC (239)
GT-AG (240)
AJ223473
M74161
Inositol polyphosphate
5-phosphatase (5ptase)
GT-AG (321)
AT-AG (322)
The differences observed between the length distribution of
U12- and U2-dependent introns raise interesting questions
about the two splicing mechanisms. In particular the accurate
pairing of donor and acceptor sites is thought to occur by two
different models in higher eukaryotes, an intron definition
model, which functions in the excision of small introns (25),
and an exon definition model, which functions in the excision
of larger introns (26). U12-dependent introns have been shown
to participate to some degree in exon definition interactions
(27) and one possible explanation for the relative dearth of
short U12-dependent introns may be that they are recognised
exclusively in an exon-dependent fashion, eliminating any
selective benefit potentially associated with the short length of
many U2-dependent introns.
A number of the U12-dependent introns found in the human
genome occur within larger gene families, suggesting that the
intron arose originally in a single ancestral gene and was dupli-
cated along with the rest of the gene as the families grew. The
presence of U12-dependent introns in some gene families,
including the calcium and sodium voltage-gated cation chan-
nels (8), the matrilin family (28), the protein kinase super-
family (7) and the E2F transcription factor family, has been
well studied. Our results found conservation of U12-dependent
introns in the phospholipase C family, the transportin family,
the diaphanous family and the cAMP-binding guanine nucle-
otide exchange factor family (see Supplementary Material) in
addition to these previously identified gene families. Addition-
ally, U12-dependent intron containing genes seem to be over-
represented in the ras-raf signal transduction pathway,
although further work is required to determine the significance
of this observation.
The observation of alternative splicing of U12-dependent
introns poses interesting evolutionary questions as well. If
U12-dependent introns convert to U2-dependent over evolu-
tionary time by accumulation of mutations at the splicing junc-
tions as previously postulated (7,9), how would this work for
alternatively spliced introns? In the case of an intron truncation
event where two different acceptors could pair with a single
donor, the intron conversion process might necessitate either
the seemingly unlikely simultaneous conversion of multiple
intron junctions or the loss of one of the splicing alternatives.
This scenario suggests that alternatively spliced U12-
dependent introns would be preferentially preserved, but is in
conflict with the observation that alternative splicing is rarer at
U12-dependent introns. A possible explanation may be that the
U12 spliceosome is less amenable to the complex regulation
patterns that alternative splicing requires and that alternative
splicing, therefore, arises less frequently at U12-dependent
WDR10p-L (WDR10)
Diaphanous 1 (HDIA1)
GT-AG (235)
GT-AG (236)
AF244931
AF051782
AF047338
AF242523
AD001528
L33798
AT-AC (81)
AT-AC (82)
Erythroid K:Cl cotransporter
(KCC1)
GT-AG (243)
GT-AG (244)
Hypothetical transmembrane
protein SBBI53
GT-AG (381)
GT-AG (382)
Spermidine
aminopropyltransferase
AT-AC (312)
GT-AG (313)
Dihydropyridine-sensitive
L-type calcium channel
alpha-1 subunit CACNL1A3
(CACNA1S)
GT-AG (9)
GT-AG (10)
Hypothetical protein
FLJ22028
GT-AG (105)
AT-AG (107)
AV725561
L26339
Autoantigen
KIAA0136 gene
Histidase
GT-AG (245)
GT-AG (246)
AT-AC (344)
GT-AG (345)
D50926
GT-AG (98)
GT-AG (99)
D16626
ERCC5 excision repair
protein (XPG)
GT-AG (212)
AT-AT (213)
L20046
KIAA1176 protein
GT-AG (188)
GT-AG (189)
AB033002
AF030409
Sodium-hydrogen
exchanger 6 (NHE-6)
AT-AC (401)
GT-AG (302)
GT-AG (303)
Sixteen genes with at least two U12-dependent introns are shown. For each
U12-dependent intron in the specified gene, the terminal dinucleotides and ID
(matching the complete intron list provided as Supplementary Material) are
provided. The accession number of a confirming expressed sequence is
provided for each gene.

Nucleic Acids Research, 2001, Vol. 29, No. 19 4013
8. Wu,Q. and Krainer,A.R. (1999) AT-AC pre-mRNA splicing mechanisms
and conservation of minor introns in voltage-gated ion channel genes.
Mol. Cell. Biol., 19, 3225–3236.
9. Dietrich,R.C., Incorvaia,R. and Padgett,R.A. (1997) Terminal intron
dinucleotide sequences do not distinguish between U2- and U12-
dependent introns. Mol. Cell, 1, 151–160.
10. Tarn,W.Y. and Steitz,J.A. (1996) Highly diverged U4 and U6 small
nuclear RNAs required for splicing rare AT-AC introns. Science, 27,
1824–1832.
11. Will,C.L., Schneider,C., Reed,R. and Lührmann,R. (1999) Identification
of both shared and distinct proteins in the major and minor spliceosomes.
Science, 284, 2003–2005.
12. Luo,H.R., Moreau,G.A, Levin,N. and Moore,M.J. (1999) The human
Prp8 protein is a component of both U2- and U12-dependent
spliceosomes. RNA, 5, 893–908.
13. Hastings,M.L. and Krainer,A.R. (2001) Functions of SR proteins in the
U12-dependent AT-AC pre-mRNA splicing pathway. RNA, 7, 471–482.
14. Shukla,G.C. and Padgett,R.A. (2001) The intramolecular stem–loop
structure of U6 snRNA can functionally replace the U6atac snRNA
stem–loop. RNA, 7, 94–105.
introns. Identification of additional examples of U12-dependent
alternative splicing and comparative analysis of gene
structures may present the most direct way towards an under-
standing of these phenomena.
Although little is known about error rates of U2-dependent
intron splicing, the calculation of a preliminary error rate for
U12-dependent splicing presents some interesting possibili-
ties. In particular, if errors occur with a significantly higher
frequency at U12-dependent introns than at U2-dependent
introns, this may point to a reason that U12-dependent introns
seem to be selected against during evolution and even are
found to be lacking entirely from some eukaryotes, such as
Caenorhabditis elegans.
In addition to the observations made here, we hope the set of
U12-dependent introns generated by this analysis will provide
a useful resource for future examinations of the minor spliceo-
some and its evolution.
15. Staden,R. (1984) Computer methods to locate signals in nucleic acid
sequences. Nucleic Acids Res., 12, 505–519.
16. Baker,W., van den Broek,A., Camon,E., Hingamp,P., Sterk,P.,
Stoesser,G. and Tuli,M.A. (2000) The EMBL nucleotide sequence
database. Nucleic Acids Res., 28, 19–23.
SUPPLEMENTARY MATERIAL
17. Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) dbEST-database
for ‘expressed sequence tags’. Nature Genet., 4, 332–333.
18. Ning,Z., Cox,A.J. and Mullikin,J.C. (2001) SSAHA: a fast search method
for large DNA databases. Genome Res., in press
19. Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W.
and Lipman,D.J. (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res., 25,
3389–3402.
Supplementary Material is available at NAR Online.
ACKNOWLEGDEMENTS
We thank M. Clamp for providing the Ensembl intron length
data and the two anonymous reviewers for many helpful
suggestions. A.L. is a Winston Churchill Scholar. R.D. is
supported by the Wellcome Trust.
20. Burset,M., Seledtsov,I.A. and Solovyev,V.V. (2000) Analysis of
canonical and non-canonical splice sites in mammalian genomes.
Nucleic Acids Res., 28, 4364–4375.
21. Dietrich,R.C., Peris,M.J., Seyboldt,A.S. and Padgett,R.A. (2001) Role of
the 3′ splice site in U12-dependent intron splicing. Mol. Cell. Biol., 21,
1942–1952.
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