Open Journal of Genetics, 2012, 2, 163-166 OJGen
http://dx.doi.org/10.4236/ojgen.2012.23021 Published Online September 2012 (http://www.SciRP.org/journal/ojgen/)
Universal RNA editing in a human liver at the fetal st age
Dong Liu1, Cong Liu1, Xiyin Wang1, Sigurdur Ingvarsson2, Huiping Chen1
1Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2Institute for Experimental Pathology and Faculty of Medicine, University of Iceland, Keldur, Iceland
Received 14 May 2012; revised 17 June 2012; accepted 10 July 2012
ABSTRACT
It is known that RNA editing occurs in human cells,
which can change the information transmission from
DNA to RNA and proteins. Most previous studies
have focused on editing of the mRNAs. Here we re-
ported that several kinds of RNAs, including miRNA,
rRNA, mRNA, miscRNA and unknown RNA, exhib-
ited base editing in a human fetal liver. Several edit-
ing types are displayed. Our data reveals that RNA
editing may occur in different species of RNAs.
Keywords: RNA Editing; miRNA; rRNA; mRNA;
miscRNA; Fetal Liver
1. INTRODUCTION
The central dogma of molecular biology is that “DNA
makes RNA makes protein” or “RNA makes DNA makes
RNA makes protein”. The transmission of information is
from DNA to RNA to protein, or from RNA to DNA to
RNA to protein. The genetic information comes from the
DNA or RNA. However, the genetic information can be
changed during the transmission. Previous studies
showed that RNA can be modified or changed, known as
RNA editing [1,2]. In addition to mRNA editing, mi-
RNAs also exhibited editing [3-5]. The main editing type
is adenosine (A) to inosine (I) modification [6]. Because
inosine acts as guanosine during translation, A-to-I con-
version in coding sequences leads to amino acid changes
and often entails changes in protein function [3,6]. RNA
can also be edited by deaminating cytidine (C) to uridine
(U).
Although the editing of mRNA includes well defined
alteration of coding capacity, the editing of regulatory
RNA molecules is less understood, but might interfere
with their functionality. A recent understanding is that
there is a broader role of RNA molecules than previously
considered and that they are crucial contributors to cel-
lular processes such as gene expression on transcriptional
and post-transcriptional levels. Editing on those regula-
tory RNA molecules could modulate how effective they
are in cellular processes.
2. MATERIAL AND METHODS
We speculate that RNA modification or editing takes
place in all kinds of RNAs. To test this hypothesis, a
liver tissue was obtained from a female fetus of 27 weeks
delivered due to severe syndrome of high blood pressure
of the mother in Tongji Hospital, Wuhan, Hubei, China.
The fetus died shortly after delivery. The mother of the
fetus had no other diseases than high blood pressure.
Small RNA (≤200 nt) was isolated from about 50 mg of
the liver tissue using a mirVanaTM miRNA isolation kit
(Ambion, Austin, TX) following the manufacturer’s in-
structions.
About 1 μg of the isolated RNAs were polyadenylated
using poly(A) polymerase (New England Biolabs). Then
the Poly(A)-tailed small RNA was purified through
phenol/chloroform extraction and ethanol precipitation.
A 5’ linker (5’-GGA CAC UGA CAU GGA CUG AAG
GAG UAG AAA-3’) was ligated to the poly(A)-tailed
RNA using T4 RNA ligase (New England Biolabs) and
the ligation products were recovered by phenol/ chloro-
form extraction followed by ethanol precipitation. Re-
verse transcription was conducted using the entire
poly(A)-tailed RNA with primer (5’-CGC TAC GTA
ACG GCA TGA CAG TG (T)24-3’) and SuperScript III
reverse transcriptase (Invitrogen) according to the manu-
facturer’s instructions. The cDNA was PCR amplified
using primers 5’-GGA CAC TGA CAT GGA CTG AAG
GAG TA-3’ and 5’-CGC TAC GTA ACG GCA TGA
CAG TG-3’. After agarose gel electrophoresis and Gold
View staining slices with PCR products were excised
and purified, using EasyPure quick gel extraction kit
(TransGen Biotech). The DNA fragments were directly
cloned into pEASY-T1 vector (TransGen Biotech). Col-
ony PCR was performed and the clones with PCR prod-
ucts were sequenced bidirectionally to avoid sequen-
cing errors. The small RNA sequences were analyzed
using BLAST analysis against the human genome and
transcripts databases and miRBase (Figure 1).

D. Liu et al. / Open Journal of Genetics 2 (2012) 163-166 164
Figure 1. Workflow of the experiments performed.
3. RESULTS AND DISCUSSION
Several kinds of cellular RNA fragments were detected.
The majority were miRNAs. Slightly more than 20% of
the cloned RNAs were degraded products of abundant
RNAs such as rRNA and mRNA, and few clones were
unknown RNAs. RNA fragments with 1 to 2 mismatches
to the sequences in the above databases were selected for
SNP search. RNA editing was suggested if the possibility
of being a SNP was excluded by the SNP databases
search. We identified 10 changes where the RNA se-
quence did not match those of the corresponding DNA
sequences. These changes were found in 4 miRNAs. In
addition to miRNAs, 28S rRNA, miscRNA (miscellane-
ous RNA, non-coding RNA), mRNA of SCL6A20 (Sol-
ute carrier family 6, member 20) gene, and unknown
RNA were scored to have RNA editing. We then de-
signed primers (Table 1) to amplify the DNA regions
that are transcribed to the RNAs above. DNA was iso-
lated from the liver tissue of the female fetus. Then PCR
products were bidirectionally sequenced using the ABI
sequencer (Figure 1). Differences between DNAs and
RNAs were identified in Figure 2 and Table 2. The wild
types of some RNAs (hsa-mir-122, hsa-mir-451 and 28S
rRNA) were also identified, suggesting that a subset of
RNAs, but not all, were edited. The hsa-mir-451 and an
unknown RNA showed two types of editing respectively,
indicating that there are different types of editing for one
kind of RNAs. We observed six possible categories of
base differences between RNA and its corresponding
DNA. In addition to A to G changes, T to C, A to C, C to
A, C to T, and G to T were also detected. There are 3
A-to-G transitions, which can be the result of deamina-
tion by ADAR or similar enzymatic activity [7]. There
are 1 C-to-T transitions, which could be mediated by
APOBEC or another deaminase. The 6 transversions
detected cannot result from deaminase-mediated editing
and the mechanism by which these differences between
RNA and DNA sequences could arise is unknown.
miRNA editing may occur in the fetal stage of human
liver, and additional species of RNAs could be edited.
Moreover, there are several types of RNA editing em-
erged. Our findings support previous results on DNA-
RNA differences in the human genome, show that these
differences include miRNAs and that they are beyond
A-to-G transitions. Our findings also suggest the exis-
tence of a new mechanism of RNA editing. Editing of
regulatory RNA molecules might interfere with their
mediated control of gene expression. Since one miRNA
has the potential to regulate several genes, the editing of
it might have significant contribution to the transcrip-
tome and proteome composition. This means that the
biological significance of RNA editing is broader that
previously considered. The universal RNA editing may
play an important role in growth and development of the
fetal livers.
4. ETHICS STATEMENT
This research has been approved by the review board of
Huazhong University of Science and Technology. We
obtained tissue samples with written informed consent
from the participants and parents/guardians of partici-
pants involved in the study. The ethics committee of
Huazhong University of Science and Technology spe-
ifically approved the procedures. c
Table 1. Primers for DNA amplification and sequencing.
Name Primer Forward Primer Reverse Annealing Temperature (˚C)
hsa-mir-23a CCTGCTCACAAGCAGCTAAG CTCTCTCTTTCTCCCCTCCAG 56
hsa-mir-99a TATTAATAGGGGGCCCATGC ATTGTTGAACGGCACTGTGT 54
hsa-mir-122 CCCGTGATGCTTCTTTTCTC AAAGCAAACGATGCCAAGAC 55
hsa-mir-451 GCCTTGTTTGAGCTGGAGTC GAGCCTGACAAGGAGGACAG 55
28s rRNA GGCGCTAAACCATTCGTAGA AGGAAGACGAACGGAAGGAC 55
miscRNA CACTGAACATGGCTTTGGTG GGTTGAGGCGTCTGTCCTAA 57
SCL6A20 mRNA ATAGGAGAAAACCGCCCTGT CTGTCTTAGCGAGGGGAGTG 55
unknown RNA GGCAGCAGGTGAGAGAGAGT GCTCTGTCTCCACCCAAATC 61

D. Liu et al. / Open Journal of Genetics 2 (2012) 163-166 165
Figure 2. Comparison of sequences of DNAs and RNAs. Arrows point to base changes between DNAs and RNAs. RE, RNA ed-
iting; WT, wild type.
Table 2. RNA editing in different species of RNAs.
RNA Sequence Type
hsa-mir-23a gene ATCACATTGCCAGGGATTTCC
hsa-mir-23a(RE) ATCACATTGCCAGGGATTCCC T to C
hsa-mir-99a gene AACCCGTAGATCCGATCTTGTG
hsa-mir-99a(RE) GACCCGTAGATCCGATCTTGTG A to G
hsa-mir-122 gene TGGAGTGTGACAATGGTGTTTG
hsa-mir-122(WT) TGGAGTGTGACAATGGTGTTTG
hsa-mir-122(RE) TGGAGTGTGACAATGGTGTTCG
T to C
hsa-mir-451 gene AAACCGTTACCATTACTGAGTT
hsa-mir-451(WT) AAACCGTTACCATTACTGAGTT
hsa-mir-451(RE)1 GAACCGTTACCATTACTGAGTT
hsa-mir-451(RE)2 ACACCGTTACCATTACTGAGTT
A to G
A to C
28s rRNA gene GCTGCGATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGT
28S rRNA (WT) GCTGCGATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGT
28S rRNA (RE) GCTGCGATCTATTGAGAGTCAGCCCTCGACACAAGGGTTTGT
A to G
miscRNA gene TCCCTGGTGGTCTAGTGGCTAGGATTCGGCGCT
miscRNA (RE) TCCCTGGTGGTCTAGTGGTTAGGATTCGGCGCT C to T
SCL6A20 gene TATTGCTGCCAGCATATC
SCL6A20 mRNA (RE) CATTGCTGCCAGCATATC T to C
unknown RNA gene GCATGGGTGGTTCAGTGGCAGAATTCTC
unknown RNA (RE)1 GCATGGGTGGTTTAGTGGCAGAATTCTC
unknown RNA (RE)2 GCATTGGTGGTTTAGTGGCAGAATTCTC
G to T
C to T

D. Liu et al. / Open Journal of Genetics 2 (2012) 163-166 166
5. ACKNOWLEDGEMENTS
This study was supported by the Huazhong University of Science and
Technology (2010MS034).
REFERENCES
[1] Bass, B.L. (2002) RNA editing by adenosine deaminases
that act on RNA. Annual Review of Biochemistry, 71,
817-846.
doi:10.1146/annurev.biochem.71.110601.135501
[2] Maas, S., Rich, A. and Nishikura, K. (2003) A-to-I RNA
editing: Recent news and residual mysteries. Journal of
Biological Chemistry, 278, 1391-1394.
doi:10.1074/jbc.R200025200
[3] Daniel, J.L., Henry, M., Nicholas J.V. and Stefan, M.
(2004). RNA editing of a miRNA precursor. RNA, 10,
1174-1177. doi:10.1261/rna.7350304
[4] Morse, D.P., Aruscavage, P.J. and Bass, B.L. (2002)
RNA hairpins in noncoding regions of human brain and
Caenorhabditis elegans mRNA are edited by adenosine
deaminases that act on RNA. Proceedings of the National
Academy of Sciences, 99, 7906-7911.
[5] Rueter, S.M., Dawson, T.R. and Emeson, R.B. (1999)
Regulation of alternative splicing by RNA editing. Na-
ture, 399, 75-80. doi:10.1038/19992
[6] Li, M.L., Isabel, X.W., Yun, L., et al. (2011) Wide spread
RNA and DNA sequence differences in the human tran-
scriptome. Science, 333, 53-58.
doi:10.1126/science.1207018
[7] Heale, B.S., Keegan, L.P., McGurk, L., et al. (2009) Ed-
iting independent effects of ADARs on the miRNA/
siRNA pathways. EMBO Journal, 28, 3145-3146.
doi:10.1038/emboj.2009.244