/ www.sciencexpress.org / 19 May 2011 / Page 1 / 10.1126/science.1207018
The transmission of information from DNA to RNA is a
critical process. We compared RNA sequences from
human B cells of 27 individuals to the corresponding DNA
sequences from the same individuals and uncovered more
than 10,000 exonic sites where the RNA sequences do not
match that of the DNA. All 12 possible categories of
discordances were observed. These differences were
nonrandom as many sites were found in multiple
individuals and in different cell types including primary
skin cells and brain tissues. Using mass spectrometry, we
detected peptides that are translated from the discordant
RNA sequences and thus do not correspond exactly to the
DNA sequences. These widespread RNA-DNA differences
in the human transcriptome provide a yet unexplored
aspect of genome variation.
DNA carries genetic information that is passed onto
messenger RNA (mRNA) and proteins that perform cellular
functions and it is assumed that the sequence of mRNA
reflects that of the DNA. This assumed precision is important
since mRNA serves as the template for protein synthesis.
Hence, genetic studies have mostly focused on DNA
sequence polymorphism as the basis of individual differences
in disease susceptibility. Studies of mRNA and proteins
analyze their expression and not sequence differences among
There are, however, known exceptions to the one-to-one
relationship between DNA and mRNA sequences. These
include errors in transcription (1, 2) and RNA-DNA
differences that result from RNA editing (3–7). Errors are
rare since proof-reading and repair mechanisms ensure the
fidelity of transcription (8–10). RNA editing is carried out by
enzymes that target mRNA post-transcriptionally: ADARs
that deaminate adenosine to inosine which is then recognized
by the translation machineries as a guanosine (A-to-G), and
APOBECs which edit cytidine to uridine (C-to-U).
Previously, sequence comparisons and computational
predictions have identified many A-to-G editing sites (6, 7,
11–13). In contrast, C-to-U changes are rare; apolipoprotein
B is one of a small number of known target genes of human
APOBEC1 (14, 15).
We obtained sequences of DNA and RNA samples from
immortalized B-cells of 27 unrelated Centre d’Etude du
Polymorphisme Humain (CEPH) (16) individuals, who are
part of the International HapMap (17, 18) and the 1000
Genomes (19) Projects. When we compared the DNA and
RNA sequences of the same individuals, we found 28,766
events at over 10,000 exonic sites that differ between the
RNA and the corresponding DNA sequences. Each of these
differences were observed in at least two individuals; many of
these were seen in B-cells, as well as in primary skin cells
and brain tissues from a separate set of individuals and in
expressed sequence tags from cDNA libraries of various cell
types. About 43% of the differences are transversions and
therefore cannot be the result of typical deaminase-mediated
RNA editing. By mass spectrometry, we also found peptide
sequences that correspond to the RNA variant sequences, but
not the DNA sequences, suggesting that the RNA forms are
translated into proteins.
We compared the DNA and RNA sequences from B-cells
of 27 unrelated CEPH individuals (table S1). We chose these
samples because much information is available on them
including dense DNA genotypes obtained using different
technologies (20, 21). The genomes of B-cells from the
CEPH collection are stable as evidenced by Mendelian
inheritance of genetic loci that allowed the construction of
Widespread RNA and DNA Sequence Differences in the Human Transcriptome
Mingyao Li,1* Isabel X. Wang,8* Yun Li,6,7 Alan Bruzel,8 Allison L. Richards,4 Jonathan M. Toung,5 Vivian G. Cheung2,3,8†
1Departments of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
2Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 3Department of
Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 4Cell and Molecular Biology
Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 5Genomics and
Computational Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
6Departments of Genetics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. 7Department of
Biostatistics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. 8Howard Hughes Medical
Institute, Chevy Chase, MD 20815, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: email@example.com
on May 19, 2011
microsatellite- to SNP-based human genetic maps (20, 21).
More recently the International HapMap Consortium (17, 18)
obtained millions of SNP genotypes and the 1000 Genomes
Project (19) sequenced the DNA of these individuals.
Comparison of sequence data from these two projects showed
high concordance (~99%). Here, we used the DNA genotypes
and sequences from the two projects for our analyses. First,
we considered sites that are monomorphic in the human
genome. A monomorphic site is one where there is no
evidence for sequence variation at that locus in dbSNP, the
HapMap and the 1000 Genomes Projects. Different studies
have analyzed these 27 and hundreds of additional individuals
for DNA variants; thus if a site has not been identified as
polymorphic, most likely all individuals have the same
sequences at these sites. But to be certain, for these sites in
the 27 individuals, we compared their DNA sequences from
the 1000 Genomes Project with the sequences of the human
reference genome and carried out traditional Sanger
sequencing (22). To be included in our analysis, we required
that each site to be covered by at least four reads in the 1000
Genomes Project and that the sequences from 1000 Genomes
are the same as the reference genome. To ensure the integrity
of the aliquots of B-cells we used for analyses, we carried out
Sanger sequencing of their DNA and found perfect
concordance of sequences with data from 1000 Genomes
(thus also the reference genome sequences) (table S2).
Second, we considered SNPs. For each individual, a SNP
locus is included only if it is homozygous and the HapMap as
well as the 1000 Genomes projects reported the same
sequence. We have high confidence in those sequences since
despite using different technologies (microarray-based
genotyping in HapMap and high-throughput sequencing in
1000 Genomes), identical sequences were obtained in the two
We sequenced the RNA of B-cells from the same 27
individuals using high-throughput sequencing technology
from Illumina (23). The resulting RNA sequence reads were
mapped to the Gencode genes (24) in the reference human
genome. In total, we generated ~1.1 billion reads of 50 base
pairs (bp) (~41 million reads and 2 Gb sequence per
individual), of which ~69% of the reads mapped uniquely to
the transcriptome (see Methods in (39)). To be confident of
the base calls, for each individual, we focused our analysis on
high quality reads (quality score ≥ 25) and sites that are
covered by at least 10 uniquely mapped reads. Another study
(25) had carried out RNA-Sequencing of the same individuals
but at a lower coverage; at these sites we compared our
sequences with those from their study, the concordance rate
of the sequences is >99.5%. This is reassuring given that the
samples were prepared and sequenced in different
/ www.sciencexpress.org / 19 May 2011 / Page 2 / 10.1126/science.1207018
For each of 27 individuals, we compared the mRNA
sequences from B-cells with the corresponding DNA
sequences (fig. S1). The comparison revealed many sites
where the mRNA sequences differ from the corresponding
DNA sequences of the same individual. To ensure that these
are actual differences and to minimize the chance of
sequencing errors, we required that at least 10% of the reads
covering a site to be different from the DNA sequence and at
least two individuals show the same RNA-DNA difference at
the site. We call each occurrence of a difference between
RNA and DNA sequences, an event, and the chromosomal
location where such a difference occurs, a site. Each person
can contribute an event to the site, thus there could be
multiple events at a site.
Among our 27 subjects, we identified 28,766 events where
the RNA sequences do not match those of the corresponding
DNA sequences. These events are found in 10,210 exonic
sites (table S10) in the human genome and reside in 4,741
known genes (36% of 13,214 genes that are covered by 10 or
more RNA-Seq reads in at least one part of the gene, in two
or more individuals). With gene orientation information in
Gencode, we observed all 12 possible categories of base
differences between RNA and its corresponding DNA (Fig.
1A). All 12 types of differences were found in each of the 27
samples; the relative proportion of each type is similar across
individuals. There are 6,698 A-to-G events which can be the
result of deamination by ADAR. There are 1,220 C-to-T
differences which can also be mediated by a deaminase.
However it is important to note that APOBEC1 and its
complementation factor A1CF that deaminate cytidine are not
expressed in our B-cells (FPKM (26) ~ 0 for both genes) thus
it is likely that an unknown deaminase or other mechanism is
involved. Even for relatively well characterized proteins such
as APOBEC1, a recent RNA-Seq study of Apobec1−/− mice
uncovered many previously unknown targets (27). In
addition, we found 12,507 transversions (43%); which cannot
result from classic deaminase-mediated editing. Since we do
not know the mechanism by which these differences between
RNA and DNA sequences arise, we refer to them as RNA-
DNA Differences or RDD. An example of RDD is a C-to-A
difference on chromosome 12 (at position 54,841,626 bp) in
the myosin light chain gene MYL6 where 16 of our subjects
have C/C in their DNA but A/C in their RNA sequences.
Another example is an A-to-C difference on chromosome 6
(at position 44,328,823 bp) in the gene, HSP90AB1, that
encodes a heat shock protein, where 8 individuals who have
homozygous A/A DNA genotype but have A/C in their RNA.
Additional examples are shown in Table 1. These sites where
RNA sequences differ from the corresponding DNA
sequences appear to be non-random since the identical
differences were found in multiple individuals: 8,163 (80.0%)
of the sites were found in at least 50% of the informative
on May 19, 2011
individuals (i.e. with RNA-Seq coverage ≥10 and DNA-Seq
coverage ≥4 at the site). Some sites were found in all or
nearly all informative individuals. For example, the DNA
sequences of all 19 informative individuals at position
49,369,615 bp of chromosome 3 in the GPX1 gene are G/G
whereas their RNA sequences are G/A. (The remaining
individuals were not included because available data did not
meet our inclusion criteria; although the data suggest the
same RDD in all remaining individuals: G/G in DNA, and
G/A in RNA).
Computational and experimental validations also upheld
these observed RNA-DNA differences. First, for 120 sites (10
sites per RDD type; randomly selected and all examples cited
in this paper; see Table 1 and table S3), we looked for
evidence of RDD in the human EST database by BLAST
alignment (28) and manual inspection of each result. For 81
of the 120 sites, we found EST clones that contain the RDD
alleles. The numbers of sites found in human ESTs are
similar across different RDD types (average 67.5%, range: 60
to 90%). Second, we examined previously identified A-to-G
editing sites (6). Fourteen of the A-to-G sites that we
identified were found in their data despite the fact that
different cell types were studied. Even the levels of editing at
these sites are similar between the two studies (see fig. S2).
Twelve additional sites were found in both studies but were
filtered because they did not meet our selection criteria.
Next, we validated our findings experimentally by Sanger
sequencing of both DNA and RNA at 12 randomly selected
sites in B-cells (2 to 9 individuals/ site), primary skin
(foreskin; 8 to 10 individuals/ site) and brain cortex (6 to 10
individuals/ site). We regrew the B-cells from our subjects
and extracted DNA and mRNA from the same aliquots of
cells. From sequencing the paired DNA and RNA samples
and analysis of each chromatogram by two individuals
independently, we confirmed 57 events in 11 sites (see Table
2, fig. S3). In EIF2AK2, in all the 8 individuals whose
samples were sequenced, three sites were found within 10
nucleotides (see below). RDD was not found in one site in
NDUFC2. Sanger sequencing is not very sensitive or
quantitative thus we do not expect to validate all sites,
especially those with low levels of RDD.
To assess if RDD shows cell type specificity, we looked
for evidence of RNA-DNA sequence differences using
primary human cells. We studied the same sites as above by
Sanger sequencing of DNA and RNA samples from primary
skin fibroblasts and brain (cortex) of a separate set of normal
individuals (for each site, we examined the DNA and RNA of
6 to 10 samples per cell type). We identified 55 RDD events
in primary skin cells and 62 events in brain cortex (Table 2).
The results suggest that most sites are shared across cell types
(Table 2); although there are exceptions, for example, an A-
to-G difference in EIF2AK2 (chr2: 37,181,512) which was
/ www.sciencexpress.org / 19 May 2011 / Page 3 / 10.1126/science.1207018
only found in B-cells and brain cortex but not in primary skin
cells. We also queried the EST database for evidence of RDD
(Table 1, table S3). The RNA alleles are seen in a wide range
of tissues from embryonic stem cells to brain and testis; they
are also found in tumors such as lung carcinoma and
Validation at the sequence level is important but does not
address all concerns such as the difficulty in aligning
sequences that are highly similar and errors introduced by
enzymes in reverse transcription steps. We believe that such
artifacts are unlikely considering the consistent patterns
across sequencing methods and the fact that we observed all
12 types of nucleotide differences. An alternate and
independent validation would be to ask whether the RNA
variants in RDD sites are translated to proteins. To do so, first
we searched mass spectrometry data from human ovarian
cancer cells (29) and leukemic cells for putative RDD sites.
Since the levels of most RDDs are less than 100%, both DNA
and the RDD-forms of the mRNAs should be available to be
translated (from here-on, we refer to mRNAs that correspond
identically to the DNA sequences as DNA-forms and those
that contain a RDD as RNA-forms). In the ovarian cancer and
leukemic cells, we indeed found examples of proteins with
peptides encoded by both DNA and RNA forms of mRNA
(table S4). Encouraged by the search results and cognizant of
possible genome instability and thus DNA mutations in
cancer cells, we carried out mass spectrometry analysis of our
We analyzed the proteome of our B-cells using liquid
chromatography-tandem mass spectrometry and detected
peptides for 3,217 proteins. Despite advances in mass
spectrometry, far less than 50% of peptides can be detected in
most studies (30, 31). We identified 327 peptides that cover
RDD sites: 299 of them are encoded by the DNA-forms and
28 by RNA-forms of RDD containing mRNAs (FDR<1%;
tables S5 and S9). For 17 RDD sites, peptides that correspond
to both DNA and RNA forms were identified (Table 3). By
BLAST alignment, we ensured that these 28 peptides are
unique to the genes that contain the RDD sites. In addition,
we sequenced the DNA of the B-cells used for mass
spectrometry and validated that the DNA sequences are the
same as the reference genome but differ from the RNA
sequences and thus do not encode the RNA-forms of the
peptides (table S2). It is easier to detect more abundant
proteins by mass spectrometry; for most RDD sites, the
unaltered DNA forms are more abundant than variant RNA
forms of mRNA (see below) thus it is not surprising to find
more peptides that corresponds to the DNA rather than the
RNA sequences. However, the counts of peptides
corresponding to the DNA and RNA forms of RDD sites
should not be taken as a measure of the proportions of DNA
versus RNA forms of mRNA that are translated because
on May 19, 2011
differences in the amino acid sequences of the DNA and
RNA forms of the peptides affect the ability of mass
spectrometry to detect them. In addition, when a peptide is
not detected, it does not mean that it is absent from the
proteome, it could be a result of sampling.
The proteomic data provide an independent validation that
mRNA sequences are not always identical to DNA
sequences, and demonstrate that RNA-forms of genes are
translated to proteins. They also show that there are peptides
in human cells that are not exactly encoded by the DNA
sequences. An example of a protein variant that results from
RDD is RPL28 (T-to-A, chr19: 60,590,467). The RDD led to
a loss of a STOP codon. We identified peptides
corresponding to the 55 amino acid extension of RPL28
protein in the ovarian cancer cells and in our B-cells (Fig. 2).
Previously identified cases of RNA editing leading to proteins
not encoded by genomic DNA, such as the apolipoprotein B
(3, 4), serotonin and glutamate receptors (32–34) in humans
and plant ribosomal protein S12 (35), also support our
hypothesis that RDD leads to protein isoforms that do not
correspond to the DNA sequences of the encoding genes.
Using our selection criteria, we found that in each person
among the Gencode genes, there are on average 1,065 exonic
events that differ in the RNA and DNA sequences. But the
number of events varied among individuals (range: 282 to
1,863) by up to 6-fold across our 27 subjects (Fig. 1B). The
degree of sequence coverage and sequencing errors in DNA
or RNA samples do not explain these individual differences
(39). Thus there is likely a biological basis for the individual
variation in the number of editing and RDD events. We found
no significant correlation between ADAR expression with the
number of RDDs or the numbers of A-to-G events (P>0.5).
Thus, either ADAR expression does not affect the number of
editing or RDD events, or our sample size is not sufficient to
detect the correlation.
The 10,210 sites which showed RNA and DNA sequence
differences are not evenly distributed across the genome:
chromosome 19 has the most whereas chromosome 13 has
the fewest number of sites. This pattern is observed after
correction for differences in size and gene density among
chromosomes. RDD sites are significantly (P<10-10) enriched
in genes that play a role in helicase activity, protein and
nucleotide binding (table S6).
We also noted that the 10,210 sites which showed RNA
and DNA sequence differences are not evenly distributed
within genes. About 44% (4,453 sites) of them are located in
coding exons (10% were found in the last exons), 4% (386
sites) are in the 5’ UTRs, and 39% (3,977 sites) are in the 3’
UTRs (see table S7, those remaining cannot be classified
because of differences in gene structures across isoforms).
The results suggest that there are more sites in the 3’ ends
than the 5’ ends of genes; a pattern that was also observed in
/ www.sciencexpress.org / 19 May 2011 / Page 4 / 10.1126/science.1207018
deamination-mediated RNA editing (27, 36). Seventy-one
percent of the coding sites result in non-synonymous amino
acid changes, including 2.1% that lead to the gain or loss of a
stop codon if translated into proteins. Relative to other
structural features in genes, we found 4% of RDD sites are
within 2 nucleotides of exon borders and 5% are within 30
nucleotides of poly(A) signals (table S7). Among RDD types,
the numbers of sites near splice junctions are quite similar but
the numbers near poly(A) sites are more different. C-to-A and
G-to-A differences are found more often near poly(A) sites.
Sites also tended to cluster; for example, 2,613 sites (26%)
are within 25 bp, and 1,059 sites (10%) are adjacent to each
other. Statistical analysis using a runs test supports that the
locations of the sites are not random (median P = 0.22). We
did not find obvious patterns or associations with motifs
shared across the sites, except for the A-to-G and A-to-C
differences that show a preference for a cytidine 5’ to the
adenosine; as previously observed in ADAR mediated A-to-G
changes (7, 34).
We examined the percentage of mRNAs that differs in
sequence from the corresponding DNA. For each site to
determine the RDD level, we counted the number of reads
with a different nucleotide from that in the corresponding
DNA sequence. The distribution of the level is bimodal (Fig.
1C); the average level is 20% (median = 13%). However, for
some sites, RDD was detected in nearly 100% of the RNA
sequences such as the A-to-C difference in the gene that
encodes an mRNA decapping enzyme, DCP1A
(chr3:53297343). This level is correlated with the frequency
and types of RNA-DNA differences. Sites found in more than
50% of the informative individuals tend to have higher levels
of RNA editing or RDD than other sites (P < 10-5; fig. S5).
The levels also differ across individuals. For example at a G-
to-A site in the gene RHOT1, which encodes a RAS protein
that plays a role in mitochondrial trafficking
(chr17:27526465), in one person, the level was 90% while in
another person, it was only 18%. We identified 437 sites with
10 or more informative individuals where the individuals with
the highest levels and the lowest levels differ by 2 fold or
more (range: 2 to 8.6 fold).
In conclusion, we have uncovered thousands of exonic
sites where the RNA sequences do not match those of the
DNA sequences; including transitions and transversions.
These findings challenge the long-standing belief that in the
same individuals, DNA and RNA sequences are nearly
identical. To increase the confidence in our results, we
obtained the DNA, RNA and protein sequences from
different individuals and cell types using a range of
technologies (fig. S1b). The samples included cell lines and
primary cells from healthy individuals and tumors. We used
data from public resources such as EST databases, The
HapMap and 1000 Genomes Projects as well as those that we
on May 19, 2011
generated with traditional Sanger sequencing, high-
throughput sequencing technologies and mass spectrometry.
Table 4 showed the DNA, RNA and peptide sequences at 15
confirmed sites which illustrate that the RNA and peptide
sequences are the same but differ from the corresponding
DNA sequences. The results support our observation that in
an individual, DNA and RNA sequences from the same cells
are not always identical and some of the variant RNA
sequences are translated into proteins. The consistent pattern
of the observations suggests that the RDDs have biological
significance and are not just “noise.” At nearly all RDD sites,
we observed only one RDD type across cell types and in
different individuals. If the DNA sequence is A/A, and the
RNA is A/C in one sample, in other samples, we see the same
A-to-C difference, but not other types of differences. These
results suggest that there are unknown aspects of transcription
and/or post-transcriptional processing of RNA. These
differences may now be studied along with those in other
genomes and organisms such as the mitochondrial genomes
of trypanosomes and chloroplasts of plants, where RNA
editing and modifications are relatively common (35, 36).
The underlying mechanisms for these events are largely
unknown. For most of the cases, we do not know yet whether
a different base was incorporated into the RNA during
transcription or if these events occur post-transcriptionally.
About 23% of the sites are A-to-G differences; some of these
are likely mediated by ADAR, but other, currently unknown,
mechanisms can be involved. If it is a co-transcriptional
process, then the signal can be in the DNA or the RNA such
as secondary structures or modified nucleotides. In addition,
as some of the RDDs are found near splice and poly(A) sites;
it is possible that this may be a facet of systematic RNA
processing steps such as splicing and cleavage (37, 38).
Our findings supplement previous studies demonstrating
RNA-DNA differences in the human genome, and show that
these differences go beyond A-to-G transition. These findings
impact our understanding of genetic variation; in addition to
DNA sequence variation, we identify individual variation in
RNA sequences. For monomorphic DNA sequences that
show RDD there is an overall increase in genetic variation.
Thus, this variation contributes not only to individual
variation in gene expression but also diversifies the proteome
since some identified sites lead to nonsynonymous amino
acid changes. We speculate that this RNA sequence variation
likely affects disease susceptibility and manifestations. To
date, mapping studies have focused on identifying DNA
variants as disease susceptibility alleles. Our results suggest
that the search may need to include RNA sequence variants
that are not in the DNA sequences.
/ www.sciencexpress.org / 19 May 2011 / Page 5 / 10.1126/science.1207018
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Acknowledgments: Dedicated to the memory of Dr. Tom
Kadesch who gave us important suggestions, taught us
salient and subtle points on gene expression, and inspired
us with his enthusiasm. Dr. Kadesch died during the
preparation of this manuscript. We thank Drs. D. Epstein,
H. Kazazian, D. Puppione and L. Simpson for suggestions
and discussions. We thank Drs. C. Gunter, R. Nussbaum
and J. Puck for comments on the manuscript, M. Morley
for help with data analysis, W. Ankener for sample
processing and J. Devlin for results on Sanger sequencing.
The mass spectrometry analysis was carried out at the
Wistar Proteomic Facility; we thank K. Speicher for help
and suggestions. Funded by grants from the National
Institutes of Health (to VGC and ML) and support from
the Howard Hughes Medical Institute. The RNA-Seq data
have been deposited to NCBI GEO under the accession
Supporting Online Material
Materials and Methods
Figs. S1 to S5
Tables S1 to S10
/ www.sciencexpress.org / 19 May 2011 / Page 6 / 10.1126/science.1207018
3 March 2011; accepted 10 May 2011
Published online 19 May 2011; 10.1126/science.1207018
Fig. 1. Characteristics of RDD sites. (A) Frequency of the 12
categories of RNA-DNA differences identified in B-cells of
27 normal individuals. (B) Number of RNA editing/ RDD
events in 27 normal individuals. (C) Bimodal distribution of
the levels of 28,766 RNA editing/ RDD events.
Fig. 2. Identification of peptides coded by both RNA and
DNA sequences. (A) The RNA-form of a RDD leads to loss
of a stop codon in RPL28 and extension of 55 amino acids.
Peptides detected by mass spectrometry are shown in red.
Extended protein sequence due to RDD is underlined. (B &
C) MS-MS data confirm the detection of peptides encoded by
the RDD containing RPL28 mRNA. The representative
spectra of one peptide (SLIGTASEPR) from ovarian cancer
cells (B) and cultured B-cells (C) are shown.
on May 19, 2011
Table 1. Selected examples of sites that show RNA-DNA Differences in B-cells and EST clones.
individuals†^ Gene Chr Position (bp)* Type
6 44,328,823 A-to-C 11 8 0.39 [0.15, 0.79] BQ355193 (head neck), BX413896 (B cell)
8 103,910,812 A-to-G 17 10 0.22 [0.12, 0.37] CD359333 (testis), BF475970 (prostate)
3 130,372,812 A-to-T 18 16 0.13 [0.10, 0.21] EL955109 (eye), BJ995106 (hepatoblastoma)
12 54,841,626 C-to-A 16 16 0.35 [0.12, 0.60] EC496428 (prostate), BG030232 (breast adenocarcinoma)
14 22,440,217 C-to-G 11 5 0.18 [0.11, 0.35] BQ232763 (testis, embryonic)
17 34,263,515 C-to-T 12 8 0.16 [0.10, 0.22] BP206252 (smooth muscle), CK128791 (embryonic stem
10 97,957,645 G-to-A 14 7 0.14 [0.11, 0.17] BF972964 (leiomyosarcoma), BE881159 (lung carcinoma)
17 77,117,583 G-to-C 2 2 0.26 [0.24, 0.28] AA625546 (melanocyte), AA564879 (prostate)
1 26,674,349 G-to-T 7 4 0.22 [0.14, 0.43] BX388386 (neuroblastoma), BE091398 (breast)
5 179,090,533 T-to-A 9 8 0.20 [0.13, 0,30] EL950052, DB558106
19 43,819,430 T-to-C 19 14 0.16 [0.10, 0.27] AI250201 (ovarian carcinoma), AI345393 (lung carcinoma)
5 40,871,072 T-to-G 6 6 0.27 [0.16, 0.45] CF124792 (T cell), DW459229 (liver)
* hg18 build of the human genome
† RNA-Seq ≥ 10 reads, DNA-Seq ≥ 4 reads
‡ Calculated by tallying RNA-Seq reads that contain RDD and those that do not.
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Table 2. Sanger sequencing of RDD sites.
* In all cases, matched DNA and RNA samples from the same individuals were sequenced
† Also reported by Li, Levanon et al, 2009(6). ‡ Known site that we used as positive control. ne = not expressed
(bp)# Type Location Amino acid change
B-Cells* Primary Skin Fibroblast* Brain (cortex)*
2 37,181,512 A-to-G 3’ UTR Not applicable 8 8 8 0 10 10
2 37,181,517 A-to-G 3’ UTR Not applicable 8 8 8 3 10 10
2 37,181,520 A-to-G 3’ UTR Not applicable 8 8 8 3 10 10
2 37,181,538 A-to-G 3’ UTR Not applicable 8 8 8 6 10 10
8 103,910,812 A-to-G
S to G 2 2 10 0 9 8
9 139,128,755 C-to-T
Synonymous (P) 9 2 8 1 10 0
5 64894960 G-to-A
E to K 2 2 8 8 8 8
6 32,833,537 G-to-A
G to S 2 2 10 10 ne ne
6 32,833,545 G-to-A
R to H 2 2 10 10 ne ne
6 32,833,550 C-to-T
Synonymous (I) 2 2 10 10 ne ne
20 35,580,977 A-to-G
Q to R 6 4 10 4 6 6
11 77,468,303 C-to-G
L to V 10 0 10 0 10 0
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Table 3 Peptides encoded by both DNA and RNA-forms of mRNA at RDD sites.
Protein Position (bp)# RDD AA change DNA-form† RNA-form†
AP2A2 chr11:976858 T-to-G Y-to-D YLALESMCTLASSEFSHEAVK DLALESMCTLASSEFSHEAVK
DFNA5* chr7:24705225 T-to-A L-to-Q VFPLLLCITLNGLCALGR VFPQLLCITLNGLCALGR
ENO1 chr1:8848125 T-to-C L-to-P EGLELLK EGPELLK
ENO3 chr17:4800624 T-to-G V-to-G LAQSNGWGVMVSHR LAQSNGWGGMVSHR
FABP3 chr1:31618424 T-to-A W-to-R MVDAFLGTWK MVDAFLGTR
FH* chr1:239747217 T-to-A I-to-K IEYDTFGELK KEYDTFGELK
HMGB1 chr13:29935772 T-to-A Y-to-N MSSYAFFVQTCR MSSNAFFVQTCR
NACA chr12:55392932 G-to-A D-to-N DIELVMSQANVSR NIELVMSQANVSR
NSF chr17:42161411 T-to-C V-to-A LLDYVPIGPR LLDYAPIGPR
POLR2B chr4:57567852 T-to-A L-to-Q IISDGLK IISDGQK
RAD50* chr5:131979610 T-to-G L-to-R WLQDNLTLR WRQDNLTLR
RPL12 chr9:129250509 A-to-G N-to-D HSGNITFDEIVNIAR HSGDITFDEIVNIAR
RPL32* chr3:12852658 G-to-T A-to-S AAQLAIR SAQLAIR
RPS3AP47* chr4:152243651 C-to-A T-to-K EVQTNDLK EVQKNDLK
SLC25A17 chr22:39520485 A-to-G E-to-G TTHMVLLEIIK TTHMVLLGIIK
TUBA1* chr2:219823379 A-to-G E-to-G EDMAALEK EDMAALGK
TUBB2C chr9:139257297 G-to-A G-to-D LHFFMPGFAPLTSR LHFFMPDFAPLTSR
* DNA sequences of these and others were verified by Sanger sequencing (see Table S2).
^RDD in RPL28 leads to the loss of a stop codon, the resulting additional peptides are found only in the RNA-form of the mRNA.
# hg 18 build of the human genome
† For each peptide, the amino acid that differs between the DNA and RNA forms are underlined.
on May 19, 2011
Table 4. Corresponding DNA, RNA and peptide sequences at selected sites.
RDD Gene Location DNA*† RNA†
TtoG CD22 chr19:40514815 CTG CGG ND MHLLGPWLLLR
TtoA DFNA5 chr7:24705225 CTG CAG VFPLLLCITLNGLCALGR VFPQLLCITLNGLCALGR
TtoC ENO1 chr1:8848125 CTG CCG EGLELLK EGPELLK
TtoA FH chr1:239747217 ATA AAA IEYDTFGELK KEYDTFGELK
TtoA HMGB1 chr13:29935772 TAT AAT MSSYAFFVQTCR MSSNAFFVQTCR
AtoC HMGB1 chr13:29935469 AAA AAC ND TMSAKEN
AtoC ITPR3 chr6:33755773 GAC GCC ND DGVEDHSPLMYHISLVALLAACAEGK
TtoG RAD50 chr5:131979610 CTA CGA WLQDNLTLR WRQDNLTLR
GtoT ROD1 chr9:114026264 GGA GTA ND NLFIEAVCSVK
GtoT RPL32 chr3:12852658 GCT TCT AAQLAIR SAQLAIR
AtoG RPS25P8 chr11:118393375 AAC GAC ND EVPDYK
CtoA RPS3AP47 chr4:152243651 ACA AAA EVQTNDLK EVQKNDLK
GtoT SUPT5H chr19:44655806 CAG CAT ND TPMYGSQTPLHDGSR
TtoC TOR1AIP1 chr1:178144365 TCA CCA ND QPSVLSPGYQK
AtoG TUBA1 chr2:219823379 GAG GGG EDMAALEK EDMAALGK
* DNA sequences are monomorphic according to dbSNP, 1000 Genomes and HapMap Projects; all individuals should have the reference allele. We
verified this by Sanger sequencing of the B-cells used for mass spectrometry.
† RDD sites are underlined.
LC-MS/MS = liquid chromatography and tandem mass spectrometry
ND = not detected by mass spectrometry; however this does not mean that the peptides are absent in the B-cell proteome. It is likely a result of sampling.
on May 19, 2011