한국 RNA 학회 조직

회장 진형종(수원대학교)
총무이사 이성욱(단국대학교)
기획이사 변종회(단국대학교)
재무이사 김동은(건국대학교)
학술이사 이강석(중앙대학교)

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 진행표
18:00~18:20 Opening remark

18:20~19:30 Reception
7월 9일(목)
19:30~22:00 Session I: RNA metabolism

22:00~ PI Meeting

08:00~09:00 조식

09:00~12:30 Colloquium: Young scientists

10:40~10:50 Coffee break

12:30~13:30 중식
7월 10일(금)
13:30~14:30 Session II: Industrial Perspective

14:30~15:00 사진촬영

15:00~17:30 Session III: Noncoding RNA

17:30~ 자유시간

08:00~09:00 조식

7월 11일(토) 09:00~11:30 Session IV: RNA in Biomedicine

Closing remark
11:30~11:40

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 Symposia
7월 9일(목)
Session I: RNA metabolism (좌장: 진형종)
19:30~22:00
강창원 RNA-mediated intrinsic attenuation of an intrinsic
19:30~20:00
(KAIST) terminator
이영훈
Control of RNA stability for M1 RNA biogenesis 20:00~20:30
(KAIST)
정선주 Role of β-catenin in cancer-related RNA alternative
20:30~21:00
(단국대) splicing
심해홍
Function of UAP56 in pre-mRNA splicing 21:00~21:30
(GIST)
김윤기 mRNA surveillance mechanisms: Implication of
21:30~22:00
(고려대) PNRC2 in nonsense-mediated mRNA decay
7월 10일(금)
Session II: Industrial Perspective (좌장: 이성욱)
13:30~14:30
김동호
RNAi in the drug discovery and development 13:30~14:00
(제놀루션)
박희경 The PNA microarray and antisense technology for
14:00~14:30
(파나진) miRNA-based diagnostics and therapeutics
Session III: Noncoding RNA (좌장: 김동은) 15:00~17:30
김빛내리
How to identify the targets of microRNA 15:00~15:30
(서울대)
최정균
Epigenetics and transcription elongation 15:30~16:00
(연세대)
박웅양 Integrative analysis of microRNA and mRNA profiles
16:00~16:30
(서울대) in human cancer
박종배 Post-transcriptional regulation of Spry2 by
16:30~17:00
(국립암센터) microRNA-21 triggers malignancy of human glioma
노유선 RNA quality control by nonsense-mediated mRNA
17:00~17:30
(서울대) decay in Arabidopsis
7월 11일(토)
Session IV: RNA in Biomedicine (좌장: 변종회)
09:00~11:30
이숙경 Promoter methylation regulates viral microRNA
09:00~09:30
(카톨릭대) expression in Epstein-Barr virus-infected cells
이희란 Development of a Novel Anti-Enteroviral Therapy
09:30~10:00
(울산대) Based on RNA Interference
민달희
Nanomaterials for oligonucleotide based drug delivery 10:00~10:30
(KAIST)
이동기 Toward the development of RNA-based
10:30~11:00
(성균관대) therapeutics: siRNA and aptamers
김동은 Oligonucleotides for therapeutic application in vitro:
11:00~11:30
(건국대) RNA-cleaving oligodeoxyribozyme and inhibitory RNA aptamer

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 Colloquium

7월 10일(금)
Colloquium: Young scientists (좌장: 이강석)
09:00~12:30

이학진 The role of stem 74 in Erm methylation of 23S

09:00~09:20
(수원대) rRNA
Human cytomegalovirus-encoded microRNAs inhibit
김성철
the production of optimal antigenic peptides of 09:20~09:40
(서울대)
MHC class I by targeting ERAP1
허인하 Uridylyl transferase TUT4 regulates Let-7 microRNA
09:40~10:00
(서울대) biogenesis

원유섭 Trans-splicing ribozyme as theragnostic agent

10:00~10:20
(단국대) against cancer
Title A new MIF4G domain-containing protein CTIF
김경미
directs the nuclear cap-binding protein 10:20~10:40
(고려대)
CBP80/20-dependent translation
염지현 Downregulation of RNase III activity enhances
10:50~11:10
(중앙대) adaptation of Escherichia coli cells to osmotic stress

김율 Role of C5 protein in rnpB transcription in

11:10~11:30
(KAIST) Escherichia coli

한국
Antisense Sib RNAs regulation for Ibs toxic proteins 11:30~11:50
(KAIST)

이정민
Characterization of BC200 RNA in cancer cells 11:50~12:10
(KAIST)

Pooja Dua Bulged asymmetric siRNA structure overcomes

12:10~12:30
(성균관대) conventional siRNA mediated off-target effects

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S1-1

RNA-mediated Intrinsic Attenuation of an Intrinsic Terminator

Sooncheol Lee, Huong Minh Nguyen and ChangwonKang

Department of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea

Short RNA transcripts are frequently aborted during the transcription

initiation process and released from ternary initiation complexes of RNA
polymerase in a repetitive cycle, which is called 'abortive initiation cycling'
process. From a single transcription unit, short abortive transcripts are
produced in vitro 30 to 100-fold more times than the full-length transcripts.
The abortive initiation cycling is no longer limited to an in vitro event,
because short RNA transcripts have recently been detected within E. coli.
However, no biological functions have been identified for short RNA
transcripts which are usually considered a throw-away product of transcription
initiation process. In this study, abortive initiation cycling transcripts are
shown to effectively attenuate an intrinsic terminator encoding an RNA
hairpin-oligo(U) structure. In the abortive initiation cycling process of
bacteriophage T7 RNA polymerase transcription with T7 promoter f10, short
G-rich RNA transcripts of various lengths (5'-GGGAGA...) were produced by
template-dictated transcription as well as short oligo(G) RNA of various
lengths by reiterative transcription due to slippage of RNA polymerase. Some
oligo(G) and G-rich RNA transcripts effectively attenuated T7 terminator Tf
in a trans-acting mannerby directly sequestering a C+U-rich part of the
terminator hairpin-forming RNA sequence, and consequently interfering with
formation of terminator hairpin. The attenuation strictly depended on
sequence-specific hybridization between the abortive RNA transcripts and a
critical part of the terminator RNA, and was intrinsic requiring nothing other
than a product of transcription. The strongest attenuation was observed with
abortive RNA transcripts from the f1.5 and f6.5 promoters among those from
all 17 promotersin T7 genome. Intrinsic attenuation at Tf mediated by
abortive RNA transcripts possibly accumulating within phage T7-infected E.
coli could help facilitate transcription-driven injection of T7 DNA into host E.
coli and read-through-dependent expression of promoter-less T7 genes 11 and
12 encoding tail tubular proteins during T7 infection of E. coli.

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S1-2

Control of RNA stability for M1 RNA biogenesis

Younghoon Lee, Yool Kim, Kwang-sun Kim, Jae-hyeong Ko, and Kook Han

Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea

Control of RNA stability is directly related to gene function. Although some

key steps for RNA degradation have been suggested, there is still a controversy
on the key stepsfor RNA degradation. We took an advantage of the fact that
M1 RNA is a metabolically stable RNA in order to more systematically
evaluate the key steps. We questioned why stable RNAs are stable in the cell
and how they escape from degradation. M1 RNA is a catalytic component of
RNase P, a tRNA processing enzyme, which is a ribonucleoprotein composed
of M1 RNA as a catalytic component and C5 protein as a protein cofactor.
M1 RNA is generated by 3′ processing from precursor M1 RNA (pM1 RNA),
which carries an extra stretch of 36 nucleotides at the 3′ end. However, the
biological significance of the 3′ processing reaction in bacterial metabolism
remained obscure. We show that 3′ processing of M1 RNA provides a
functional mechanism for the protection of the primary transcript against
degradation. Since this 3′ processing reaction is initiated by RNase E, this
enzyme therefore plays a role in the conversion of unstable pM1 RNA into
stable M1 RNA. In addition to pM1 RNA, large rnpB-containing transcripts are
produced from unknown upstream promoters. To examine their biological
relevance to M1 RNA biosynthesis, we constructed a model upstream transcript,
upRNA, and analyzed its cellular metabolism. We found that upRNA was
primarily degraded rather than processed to M1 RNA in the cell and that this
degradation occurred in RNase E-dependent manner. Considering that RNase E
is a processing enzyme involved in 3′ end formation of M1 RNA, our results
imply that this enzyme plays a dual role in processing and degradation to
achieve tight control of M1 RNA biosynthesis. Meanwhile, C5 protein functions
as a cofactor in the RNase P reaction. We examined whether C5 protein is
involved in maintaining metabolic stability of M1 RNA. The sequestration of
C5 protein available for M1 RNA binding reduced M1 RNA stability in vivo,
and its reduced stability was recovered via overexpression of C5 protein. These
results demonstrate that C5 protein also plays an essential role in stabilizing
M1 RNA in the cell.

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S1-3

Role of β-catenin in cancer-related RNA alternative splicing

Jung Hur, Su-ho Kim, Injoo Hwang, Hee Kyu Lee, Inae Kim and Sunjoo Jeong
National Research Lab for RNA Cell Biology, BK21 Graduate Program for RNA
Biology and Department of Molecular Biology, Dankook University,
Yongin, Gyeonggi-do 448-701, Republic of Korea
Recent findings demonstrate that multiple mRNAs are co-regulated by one or
more RNA-binding proteins (RBPs) toorchestrate their splicing, export, stability,
localization and translation. Aberrant expression of RBPs affects many steps of
RNA metabolism, significantly altering expression of many RNA transcripts.
Thus, altered expression and dysfuncting of RBPs are implicated in the
development of various diseases including cancer. Activated β-catenin regulates
the transcription of oncogenic target genes and is critical for tumorigenesis. We
have recently demonstrated that the cancer-related transcription factor β-catenin
can also bind RNA, thus acting as a RBP (Lee and Jeong, 2006).
Overexpressed β-catenin can also regulate alternative splicing of Estrogen
Receptor-β mRNA and stabilize Cycloocygenase-2 and cyclinD1 mRNAs in
colon cancer cells (Lee et al., 2006; Lee et al., 2007). To test if activated β
-catenin globally affects alternative splicing of many other mRNAs in colon
cancer cells, we performed splicing microarray from the HCT116 colorectal
cancer cells with activated form of β-catenin. We assessed the role of the β
-catenin in alternative splicing of many transcripts by using a stringent
algorithm and identified 97 exons that were differentially spliced in the β
-catenin overexpressed cells. We tested some of these regulated genes and
validated their changes in alternative splicing pattern by RT-PCR analyses. Our
results extend the previous finding that β-catenin has an important role in both
transcription and alternative splicing of many cancer-related genes.

References:
Lee HK and Jeong S (2006) β-Catenin stabilizes cyclooxygenase-2 mRNA by
interacting with AU-rich elements of 3’-UTR. Nucleic Acids Res., 34: 5705-5714.
Lee HK, Choi YS, Park YA and Jeong S (2006) Modulation of oncogenic
transcription and alternative splicing by β-catenin and an RNA aptamer in colon
cancer cells. Cancer Res., 66: 10560-10566.
Lee HK, Kwak H, Hur J, Kim IA, Yang JS, Park MW, Yu J and Jeong S (2007) β
-Catenin regulates multiple steps of RNA metabolism as revealed by the RNA
aptamer in colon cancer cells. Cancer Res., 67: 9315-9321.

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S1-4

Role of UAP56 in pre-mRNA splicing

Haihong Shen

Department of Life Science, GIST, Gwangju, Republic of Korea

The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box

protein known to promote prespliceosome assembly. Here, using a series of
hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or
dsRNA unwindase/helicase activity, we assess the relative contributions of
these biochemical functions to pre-mRNA splicing. We show that
prespliceosome assembly requires hUAP56's ATP-binding and ATPase
activities, which, unexpectedly, are required for hUAP56 to interact with
U2AF(65) and be recruited into splicing complexes. Surprisingly, we find that
hUAP56 is also required for mature spliceosome assembly, which requires, in
addition to the ATP-binding and ATPase activities, hUAP56's dsRNA
unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4
and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that
both these activities are dependent on U2AF(65). Our results indicate that
hUAP56 first interacts with U2AF(65) in an ATP-dependent manner, and
subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the
spliceosome.

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S1-5

mRNA surveillance mechanisms: Implication of PNRC2 in

nonsense-mediated mRNA decay
Yoon Ki Kim

School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA

surveillance mechanism by which aberrant mRNAs harboring premature
termination codons (PTCs) are degraded before translation. However, to date,
how NMD machinery recruits the general decay complex to faulty mRNAs
and degrades those mRNAs remains unclear. Recently, we identify human
proline-rich nuclear receptor coregulatory protein 2 (PNRC2) as a new Upf1-
and Dcp1a-interacting protein. Downregulation of PNRC2 abrogates NMD, and
artificially tethering PNRC2 downstream of a normal termination codon
reduces mRNA abundance. Accordingly, PNRC2 preferentially interacts with
hyperphosphorylated Upf1 compared with wild-type Upf1 and triggers
movement of hyperphosphorylated Upf1 into processing bodies (P-bodies). Our
observations suggest that PNRC2 plays an essential role in mammalian NMD,
mediating the interaction between the NMD machinery and the decapping
complex, so as to target the aberrant mRNA-containing RNPs into P-bodies.
In addition to NMD, other mRNA surveillance mechanisms will be discussed.

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C-1

The role of stem 74 in Erm methylation of 23S rRNA

Hak Jin Lee and Hyung Jong Jin

Department of Bioscience and Biotechnology, College of Natural Science,

The University of Suwon, Hwasung, Kyunggi-Do 445-743, Republic of Korea

Erm proteins mono- or dimethylate a specific residue (A2058) in peptidyl

transferase circle of 23S rRNA domain V to reduce the affinity of MLS
antibiotics to it, thereby conferring the resistance on the microorganisms.
ErmSF is one of Erm proteins which originates from
Streptomycesfradiae,atylosinproducerandusedinthisstudy. Domain V has been
known as a complete substrate to contain all the structural elements which
are recognized and methylated by Erm proteins. Though Kd and Km values
become higher and Vmax lowered being a less efficient substrate as the
substrate was reduced to 72nt RNA fragment consisting of stem 73 and stem
74, it still shows the activity comparable to domain V, maintaining the
structural features of the domain V encompassing stems 73 and 74 as judged
by enzyme and chemical probing. To examine the role of stem 74 in
methylation by Erm protein, gradual truncation of stem 74 was carried out to
get 53nt RNA and 41nt RNA fragment and their Kd values and kinetic
parameters was determined. Concomitant with truncation of stem 74, Kd and
Km increased accordingly as expected suggesting that it interacts with ErmSF.
Footprinting analysis confirmed many of the residues of stem 74 including
truncated residues interacted with ErmSF. In contrast to this, Vmax remains
constant even with truncation suggesting that while stem 74 engages in
recognition by Erm protein through binding to it, it is no longer bound to
the enzyme when methylated product is being released from the enzyme.
This conformational change was thought to be carried out by the enzyme
alone because the truncated RNAs still show the same properties even though
we cannot rule out the possibility that part of stem 74 has the enough
potential to carry out the conformational change.

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C-2

Human cytomegalovirus-encoded microRNAs inhibit the production

of optimal antigenic peptides of MHC class I by targeting ERAP1
Sungchul Kim, Jinwook Shin, Kwangseog Ahn

Laboratory of Antigen Presentation, School of Biological Sciences,

Seoul National University, Seoul 151-742 Republic of Korea

The human cytomegalovirus (HCMV) is a member of beta-herpesvirus

subfamily. It is a widespread pathogen which may cause chronic infection. It
is also thought to be the major infectious cause of birth defects, severe
diseases in immunocompromised individuals, and so on. MicroRNAs are a
class of small noncoding-RNAs which function as post-transcriptional genetic
regulators. Many viruses as well as eukaryotes encode microRNAs which may
participate in viral immune evasion from host immune system. Recently,
several groups identified and characterized HCMV-encoded microRNAs.
Because many genes in US2-US11 genomic region inhibits MHC class I
surface expression, we hypothesized that microRNAs in US2-US11 region of
HCMV genome have host targets which can regulate antigen presentation of
MHC class I. Using microarray analysis, we found that these microRNAs
altered the transcription pattern of some immune regulatory genes. Among
these genes, we focused endoplasmic reticulum aminopeptidase 1(ERAP1)
which enhances or limits antigen presentation of MHC class I by trimming
epitopes to 8-9 residues. We validated ERAP1 as a direct target of
hcmv-miR-US4 by quantitative real-time PCR and luciferase reporter assay.
The targeting effect of hcmv-miR-US4 to 3'UTR of ERAP1 mRNA might be
seed region binding-dependent. Thus, HCMV encodes miRNAs which can
modulate the host immune system by inhibiting of the optimizing process of
antigenic peptides in the ER.

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C-3

Uridylyl Transferase TUT4 Regulates Let-7 MicroRNA Biogenesis

Inha Heo, Young-Kook Kim, Minju Ha, Mi-Jeong Yoon, Jun Cho, Jinju Han,
Chirlmin Joo, and V. Narry Kim

Creative Research Center and School of Biological Sciences,

Seoul National University, Seoul 151-742 Republic of Korea

MicroRNAs (miRNAs) are key regulators of many cellular functions,

therefore, their biogenesis needs to be tightly controlled. Let-7 miRNAs are
repressed post-transcriptionally by Lin28, the pluripotency promoting factor, in
embryonic stem cells and liver cancer cells. Although Lin28 was reported to
mediate the repression by inducing the uridylation of precursor let-7 miRNA
(pre-let-7), the identity of the uridylating enzyme remained unknown. Here we
identify a non-canonical poly (A) polymerase, TUT4, as the uridylyl
transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 in the cytoplasm.
TUT4, in turn, adds an oligo-uridine tail to the 3’ end of pre-let-7. The
uridylating activity for pre-let-7 is uniquely observed with TUT4 among
non-canonical poly (A) polymerases. We also find that let-7 family is the
major target of TUT4 and Lin28. This study uncovers the role of TUT4 as a
post-transcriptional suppressor of let-7 miRNA biogenesis, which may have
implications for stem cell research and cancer biology.

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C-4

Trans-splicing ribozyme as theragnostic agent against cancer

You Sub Won, Sung Jin Kim, Guyee Ban, and Seong-Wook Lee

Creative Research Center and School of Biological Sciences,

Seoul National University, Seoul 151-742 Republic of Korea

The human cytomegalovirus (HCMV) is a member of beta-herpesvirus

subfamily. It is a widespread pathogen which may cause chronic infection. It
is also thought to be the major infectious cause of birth defects, severe
diseases in immunocompromised individuals, and so on. MicroRNAs are a
class of small noncoding-RNAs which function as post-transcriptional genetic
regulators. Many viruses as well as eukaryotes encode microRNAs which may
participate in viral immune evasion from host immune system. Recently,
several groups identified and characterized HCMV-encoded microRNAs.
Because many genes in US2-US11 genomic region inhibits MHC class I
surface expression, we hypothesized that microRNAs in US2-US11 region of
HCMV genome have host targets which can regulate antigen presentation of
MHC class I. Using microarray analysis, we found that these microRNAs
altered the transcription pattern of some immune regulatory genes. Among
these genes, we focused endoplasmic reticulum aminopeptidase 1(ERAP1)
which enhances or limits antigen presentation of MHC class I by trimming
epitopes to 8-9 residues. We validated ERAP1 as a direct target of
hcmv-miR-US4 by quantitative real-time PCR and luciferase reporter assay.
The targeting effect of hcmv-miR-US4 to 3'UTR of ERAP1 mRNA might be
seed region binding-dependent. Thus, HCMV encodes miRNAs which can
modulate the host immune system by inhibiting of the optimizing process of
antigenic peptides in the ER.

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C-5

A new MIF4G domain-containing protein CTIF directs the nuclear

cap-binding protein CBP80/20-dependent translation
Kyoung Mi Kim1*, Hana Cho1*, Kobong Choi2*, Jaedong Kim2, Bong-Woo Kim1,
1 2 1
Young-GyuKo , Sung Key Jang , and YoonKi Kim
1
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701,
2
Republic of Korea, Department of Life Science, Pohang University of Science and
Technology, Pohang, Kyungbuk 790-784, Republic of Korea

During mRNA export via the nuclear pore complex (NPC) in mammalian
cells, the mRNA is believed to undergo the translation mediated by nuclear
cap-binding proteins 80 and 20 (CBP80/20). Nonsense-mediated mRNA decay
(NMD), one of the best-characterized mRNA surveillance mechanisms, has
been shown to occur on CBP80/20-bound mRNAs. After CBP80/20-dependent
translation, CBP80/20 is likely to be replaced by cytoplasmic cap-binding
protein eIF4E, which directs steady-state translation. However, the underlying
molecular mechanism and even the exact identity of CBP80/20-dependent
translation still remain obscure. Here, we identify a new MIF4G
domain-containing protein, CTIF. CTIF directly interacts with CBP80 and is
in complex with the functional form of the CBP80/20-dependent translation
initiation complex. Depletion of endogenous CTIF from the in vitro translation
system blocks the translation of CBP80-bound mRNAs, in a way that is
restored by addition of purified CTIF. Accordingly, downregulation of
endogenous CTIF abrogates NMD. We also show that CTIF is localized to
the perinuclear region. Our observations demonstrate the existence of
CBP80/20-dependent translation and support the idea that CBP80/20-dependent
translation is mechanistically different from steady-state translation through
identification of a specific cellular protein, CTIF.

*These authors contributed equally to this work.

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C-6

Downregulation of RNase III activity on bdm mRNA degradation

enhances adaptation of Escherichia coli cells to osmotic stress
1* 1* 1 1
Se-Hoon Sim , Ji-Hyun Yeom , Eunkyoung Shin , Yoonsoo Hanh , Choy
2 3 3 1
Shin , Jae-Hong Kim , Jeehyeon Bae , and Kangseok Lee
1 2
Department of Life Science and Graduate School of Education, Chung-Ang
3
University, Seoul 156-756, Republic of Korea, Graduate School of Life Science and
Biotechnology, Pochon CHA University, Seongnam 463-836, Republic of Korea

During the course of experiments aimed at the identification of genes

whose expression is RNase III-dependent in Escherichia coli, we found that
the steady-state level of bdm mRNA was dependent on the cellular
concentration of RNase III. The half-life of adventitiously overexpressed bdm
mRNA and the activities of transcriptional bdm′-′cat fusion were dependent on
the cellular concentrationof RNase III, indicating the existence of cis-acting
elements in bdm mRNA reacted by RNase III. In vitro cleavage analyses of
bdm mRNA identified two RNase III cleavage motifs, one in the 5’-UTR and
the other in the coding region of bdm mRNA, and indicated RNase III
cleavages in the coding region as a rate-determining step for bdm mRNA
degradation. We further discovered that downregulation of the ribonucleolytic
activity of RNase III is required for sustainment of elevated bdm mRNA
levels induced by RcsB during osmotic stress, and that overexpression of
Bdm renders cells to more efficiently form biofilm. These findings reveal the
existence of an additional regulatory pathway in the Rcs signaling system,
which modulates bdm expression and consequently, adaptation of E. coli cells
to osmotic stress.

*These authors contributed equally to this work.

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C-7

Role of C5 protein in rnpB transcription in Escherichia coli

Yool Kim and Younghoon Lee

Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea

Escherichia coli RNase P is composed of a large RNA subunit (M1 RNA)

and a small protein subunit (C5 protein). Since both subunits are assembled
in a 1:1 ratio, expression of M1 RNA and C5 protein should be coordinately
regulated for RNase P to be efficiently synthesized in the cell. However, it is
not known yet how the coordination occurs. In this study, we investigated
how overexpression of C5 protein affects expression of the rnpB gene
encoding M1 RNA, using a lysogenic strain, which carries an rnpB-lacZ
transcription fusion. Primer extension analysis of rnpB-lacZ fusion transcripts
showed that the overexpression of C5 protein increased the amount of the
fusion transcripts, suggesting that rnpB expression increases with the increase
of intracellular level of C5 protein.

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C-8

Antisense Sib RNAs regulation for Ibs toxic proteins

Kook Han, Geunu Bak, Hongmarn Park, and Younghoon Lee

Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea

Ibs (induction brings stasis) toxic proteins are very hydrophobic 18- to 19-
amino acid proteins encoded by five highly homologous sequences in E. coli
K-12 genome.When they are overexpressed, these short hydrophobic proteins
lead to growth arrest, disturbing the membrane potential. As counterparts, Sib
(short, intergenic, abundant) antitoxin RNAs are approximately 140
nucleotidesin length transcribed from the complementary strands opposite to
each of the five ibs genes. The Sib RNAs repress the synthesis of ibs
proteins, leading to degradation of target ibs mRNAs by complementary base
paring. Interestingly, each Sib RNA uniquely represses its corresponding target
mRNA without cross-regulation, although they are extremely homologous for
each other. However, the detail understanding of the mechanisms of regulation
by base paring target mRNAs remains to be elucidated. Here, we report
sequences of Sib RNA for the regulation of ibs expression in the view of its
essentiality and specificity.

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C-9

Characterization of BC200 RNA in cancer cells

Jungmin Lee, Geunu Bak, Youngmi Kim, Kyung Hwan Kim, and Younhgoon Lee

Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea

BC200 RNA (brain cytoplasmic 200 RNA), a primate noncoding RNA

which istranscribed by RNA polymerase III, functions as a translational
modulator blocking local synaptodendritic protein synthesis in neurons of
human nervous system. BC200 RNA is not detected in somatic cells other
than neurons. However, the neuron-only expression of BC200 does not hold
true in a number of tumors (carcinomas of breast, cervix, oesophagus, lung,
ovary, parotid, and tongue). In this study, we examined biogenesis and
functions of BC200 in cancer. We showed that BC200 RNA was expressed at
high levels in several breast cancer cells, but at low levels in normal breast
cells. Primer extension analysis revealed that BC200 of breast cancer cells
had extra nucleotides at its 5' end compared to neuronal BC200, whose size
is 200 nt. Self-ligation RACE (rapid amplification of cDNA ends) showed
that BC200 had long forms in size, including precursors, suggesting that
BC200 biogenesis in cancer cells differs from that in neurons. We identified
putative binding partner proteins of BC200 for uncovering BC200 functions in
cancer cells.

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C-10

Bulged asymmetric siRNA structure overcomes conventional siRNA

mediated off-target effects

Pooja Dua

Global Research Laboratory for RNAi Medicine, Sungkyunkwan University, Suwon,

Republic of Korea

Off-target silencing is one of the major concerns of application of RNA

interference. siRNA mediated gene silencing occurs in a sequence dependent
rather than target dependent manner, hence, limited complimentarily between
the RNA duplex and the transcripts is sufficient to initiate gene silencing. To
overcome this, we have developed "Bulged siRNA", with a single nucleotide
bulge placed in the antisense strand. The bulged siRNAs showed significant
reduction in silencing of mismatched targets as compared to the unmodified
siRNAs, while silencing for the perfectly matched targets was nearly
unaffected by the structural modification. We also compared our structure with
the 2’-methoxy modified siRNAs being used currently and found that 3 out
of 4 siRNAs tested gave better discrimination of perfectly matched vs
mismatched targets with the bulged structure. Finally to reduce both the sense
and antisense strand mediated off-target effects we combined the bulged
structure with the asiRNA structure developed in our lab and found overall
reduction in siRNA off-targets. The bulged structure proposed here has
enhanced application as they can be easily translated to shRNAs or
bacterially-expressed siRNAs where chemical modification cannot be
introduced.

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S2-1

RNAi for drug target discovery

Dongho Kim

Genolution Pharmaceuticals, Inc.

Searching for novel genes in specific pathway is a routine but tedious

process unless tight controlling and sophisticated designing is established. It
can be ends up with filed-up data unless there is a following-up of functional
genomics for in vitro and in vivo validation. For the functional genomics
RNAi technology turned out to be the life saver because of high efficacy and
convenient access. We will show examples of smooth connection from gene
hunting to in vivo validation using shRNA technology in cancer models.
Several genes involved in drug resistance and cancer survival have been
sought using microarry. Using a novel shRNA synthesis technology the whole
validation for efficient siRNA selection can be very time and cost-saving step.
Furthermore, the in vitro and in vivo validation can be acceleratedby
construction of shRNA expression vector under the inducible promoter. By
adopting novel RNA transcriptional technology, screening for a hyperactive
siRNA and discovery for a novel drug target gene can be escalated. We will
present a few experiences in RNAi and its application to drug target gene
discovery as part of endeavor for new drug development.

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S2-2

The PNA microarray and antisense technology for miRNA-based

diagnostics and therapeutics
Jae-Jin Choi , Su Young Oh, Hyunsun Kim, Yeong Soon Ju and Heekyung Park

Panagene Inc., Daejeon, 305-510, Republic of Korea

MicorRNAs (miRNAs) are an abundant class of small non-coding RNAs,

typically 20-23 nucleotides in length that regulate gene silencing at the
post-transcriptional level. Furthermore, miRNAs have been implicated in viral
infections, cardiovascular disease, and muscular disorders, as well as in the
onset and progression of cancers. Identification of miRNAs and their
respective targets may provide potential diagnostic and prognostic disease
biomarkers and new therapeutic strategies to treat disease.
PNA (Peptide Nucleic Acid) is synthetic DNA analogue in which the
backbone is replaced by repetitive units of N- (2 aminoethyl) glycine. PNAs
have stronger affinity and greater specificity than DNA oligonucleotides for
binding to DNA and RNA. Also, PNAs are resistant to nuclease, which is
essential for a miRNA antisense that be exposed to abundant serum and
cellular nucleases. These properties make PNAs well suited for miRNA
TM
microarray and miRNA antisense. We developed PANArray miRNA for
TM
miRNA expression profiling and PNAs miRNA inhibitors for miRNA
antisense.
PANArrayTM miRNA is highly sensitive, specific and reproducible to
analyze miRNA expression profiles with direct miRNA labeling technique on
chip after hybridization. Unlabeled total RNA is hybridized to the PNA
microarray and then only hybridized miRNA labeling is performed by using
enzymatic ligation with pCp-Cy3. Our results showed very low cross
hybridization for miRNAs differing by single nucleotide such as human let7
(a-i) family. Also, this PNA microarray can detect small amounts of
individual miRNAs from <400 ng of total RNA with above 99% correlation
between independent replicates in 6 hrs.
The miRNA antisense is unique and effective technique for miRNA
functionalization and therapeutic targeting. We tested PNA-based antisense for
over 100 miRNAs and found that all of the PNA could act as powerful
miRNA antisense. These results demonstrated that PNA-based miRNA
antisense is a more effective than other DNA-based miRNA antisense.
These results suggest that PNA technology is a valuable and powerful tool in
miRNA-based diagnostics and therapeutics.

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S3-1

How to discover the functions of microRNAs

Seogang Hyun, Jung Hyun Lee, Hua Jin, Seongyeon Lee, JinWu Nam,
Gina Lee, Jongkyeong Chung, and V. Narry Kim

Creative Research Center and School of Biological Sciences,

Seoul National University, Seoul 151-742, Republic of Korea

microRNAs (miRNAs) are non-coding RNAs of ~22 nt, which act as

post-transcriptional repressors by base-pairing to the 3’ untranslated region
(UTR) of their cognate mRNAs. The physiological functions of individual
miRNAs remain largely unknown, and it is a daunting task to understand the
biological function of a given miRNA and to identify its physiologically
relevant targets. Studies of miRNA function rely heavily on computational
algorithms that predict target genes, but in spite of their utility, these target
prediction programs generate many false positives because regulation in vivo
depends on target message availability and complementary sequence
accessibility. To overcome the difficulties in identifying real targets, various
experimental approaches have been developed, including microarrays,
proteomic analyses, and biochemical purification of the miRNA-mRNA
complex. Genetic approaches using model organisms can also be useful tools
to study the biological roles of miRNAs at both the organismal and
molecular levels. I will discuss several different approaches that we have
taken to discover the functions of miRNAs in cell growth and death.

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S3-2

Epigenetics and transcription elongation

Jung Kyoon Choi

Yonsei Genomic Institute, Seoul, Republic of Korea 151-742

Recent epigenetic studies on a genome-wide level point out that in many

instances, the transcript body as well as the promoter can be a major target
of epigenetic mechanisms. DNA methylation, H3K36me3, and nucleosome
remodeling have been shown to take place within the transcript body, raising
a possibility that these epigenetic marks play a role other than regulating
transcription initiation, challenging the consensus view. Their suggested roles
include the regulation of RNA splicing by slowing polymerase elongation and
the suppression of aberrant transcription initiation or antisense RNA
expression within the gene body or even non-coding RNA region. In this
talk, I will present a few lines of evidence from the literature and from our
preliminary analyses that sheds new light on crosstalk between epigenetic
mechanisms and RNA processing machinery.

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S3-3

Differential microRNA expression upon ionizing radiation

in lung cancer cells
3 3 2 3
Hye-Youn Sung , Ae-Kyung Park , Sun-HyeJeong , Shun-Zi Jin ,
1 2 2,3
Hong-Gyun Wu , Ju Han Kim , Woong-Yang Park
1 2 3
Departments of Radiation Oncology, Biomedical Sciences, Biochemistry and
Molecular Biology, Seoul National University College of Medicine,
Seoul 110-799, Republic of Korea

Radiotherapy is one of the most widely used modality to treat human

cancers. Lung cancer is one of the most deadly types of cancer, and the
radiotherapy is playing a key role in the treatment of inoperable patients with
stage III non-small cell lung cancer (NSCLC). The regulatory molecules like
microRNA and transcription factors are capable of affecting expressions of a
large number of target genes in a concerted manner. We analyzed
time-dependent changes in microRNA expressions after irradiating NCI-H460
and NCI-H1299 human NSCLC cells, which are showing different sensitivity
against ionizing radiation. Large numbers of microRNA were altered by IR,
but the temporal patterns were quite different each other. let-7 family of
microRNAs can regulate the translation of oncogenes like KRAS. let-7
family of microRNAs is over-represented in the microRNAs showing altered
expression upon ionizing radiation. We also demonstrated that let-7g can
enhance the radiosensitivity. From these results we suggest that microRNAs
can be a valuable tool to enhance the efficiency of radiotherapy.

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S3-4

Post-transcriptional regulation of Spry2 by microRNA-21 triggers

malignancy of human glioma

Jong-Bae Park

Specific Organs Cancer Branch, Division of Translational & Clinical ResearchⅡ,

Research Institute, National Cancer Center,
Goyang, Gyeonggi-do 410-769, Republic of Korea.

Hyaluronan (HA), a principal glycosaminoglycan in the extracellular matrix

of brain, is a critical factor for the glioma invasion by facilitating cell
adhesion, motility and proliferation. In this study, we showed that
microRNA-21 (miR-21), a well-known oncogenic microRNA in the GBM3, is
upregulated by HA and facilitates the glioma invasion by increasing MMP-9
expression. The miR-21 increased Ras/MAPK signaling and followed MMP-9
expression by targeting Spry2. Consistently, Spry2 protein levels were
significantly decreased in 83.3% of invasive WHO grade II-IV human glioma
tissues. Furthermore, Spry2 protein level was inverse-correlated with miR-21
level but not with Spry2 mRNA level. These findings implicate the clinical
significance of post-transcriptional control of Spry2 by miR-21 in glioma
progression.

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S3-5

RNA quality control by nonsense-mediated mRNA decay

in Arabidopsis

Yoo-Sun Noh

School of Biological Sciences & Global Research Laboratory for Floral Regulatory
Signaling, Seoul National University, Seoul 151-742, Republic of Korea

SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) is regulated by a

complex transcriptional regulatory network that allows for the integration of
multiple floral regulatory inputs fromphotoperiods, gibberellin, and
FLOWERING LOCUS C. However, the posttranscriptional regulation of SOC1
has not been explored. Here we report that EARLY FLOWERING 9 (ELF9),
an Arabidopsis RNA-binding protein, directly targets the SOC1 transcript and
reduces SOC1mRNA levels, possibly through a nonsense-mediated mRNA
decay (NMD) mechanism, which leads to the degradation of abnormal
transcripts with premature translation termination codons (PTC). The fully
spliced SOC1 transcript is up-regulated in elf9 mutants as well as in mutants
of NMD core components. Further, a partially spliced SOC1 transcript
containing a PTC is up-regulated more significantlythan the fully spliced
transcript in elf9 in an ecotype-dependent manner. A Myc-tagged ELF9
protein (MycELF9) directly binds to the partially spliced SOC1 transcript.
Previously known NMD target transcripts of Arabidopsis are also up-regulated
in elf9 and recognized directly by MycELF9. SOC1transcript levels are also
increased by the inhibition of translational activity of the ribosome. Thus, the
SOC1 transcript is one of the direct targets of ELF9, which appears to be
involved in NMD-dependent mRNA quality control in Arabidopsis.

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S4-1

Promoter methylation regulates viral microRNA expression in

Epstein-Barr virus-infected cells

Do Nyun Kim, Se Mo Jun, Sang Taek Oh, Jung Seon Seo, and Suk Kyeong Lee

Research Institute of Immunobiology, Department of Biomedical Sciences, College of

Medicine, The Catholic University of Korea, Seoul, Republic of Korea

MicroRNAs (miRNAs) are 21–24 nt long small noncoding RNAs that are
involved in post-transcriptional gene regulation by binding to the 3’UTR of
target mRNAs. Epstein-Barr virus (EBV) encodes two groups of viral
miRNAs: BHRF1 miRNAs and BART miRNAs. Previous reports have shown
that BART miRNAs are expressed in the cell lines that exhibit latency I and
latency III EBV infection, whereas BHRF1 miRNAs are expressed only in
the latency III cell lines. Interestingly, differential expression level was
detected for the BART miRNAs in different tumors as well as in different
cell lines. EBV genomein latency I and II is known to contain epigenetically
silenced latent Cp/Wp promoters by DNA hypermethylation. To clarify
whether the expression of EBV miRNAs is also regulated by epigenetically,
the methylation status of the BART miRNA promoter was investigated in the
different cell lines using combined bisulfite restriction analysis (COBRA) and
pyrosequencing. A higher prevalence of the promoter methylation was
observed in the cell lines with low BART miRNA expression compared with
those with high BART miRNA expression. In addition, 5-aza-2'-deoxycytidine
(5-aza-Cd) treatment not only reduced the promoter methylation but also
enhanced BART miRNA expression. Furthermore, induction of BHRF1
miRNAs expression accompanied by the activation of Cp/Wp promoter was
detected in the latency I cell lines following 5-aza-Cd treatment. However, a
histone deacetylase (HDAC) inhibitor trichostatin A did not affect EBV
miRNAs expression alone or in combination with 5-aza-Cd. Taken together,
our results suggest that the expression of EBV miRNAs are regulated by the
DNA methylation but not by histone acetylation of the relevant promoters in
latently infected cells.

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S4-2

Development of a novel anti-enteroviral therapy

based on RNA interference

Jeonghyun Ahn, Eun Jung Jun, Hui Sun Lee, Yoo Kyum Kim, and Heuiran Lee

Department of Microbiology, Research Institute for Biomacromolecules, University of

Ulsan College of Medicine (Asan Medical Center), Seoul, Republic of Korea

Non-polio enteroviruses including coxsackievirus A and B, are associated

with many human diseases, ranging from mild aseptic meningitis to
life-threatening dilated cardiomyopathy. Yet, there have been practically no
preventive and therapeutic tools. This virus consists of a large number of
serotypes with high genetic instability mainly due to lack in proofreading
activity of virus RNA polymerase. Recently, a body of independent studies
has consistently reported the potency of "RNA interference" for inhibition of
a variety of viruses, such as HIV, RSV, and poliovirus. Based on these facts,
we previously provided solid evidence regarding the ability of small
interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3)
and to induce antiviral activity both in vitro and in vivo.
However, the antiviral potency of siRNAs is limited in these virus groups
characterized by high sequence diversity and genetic instability. To overcome
this drawback, we developed novel siRNA-designing software to readily select
siRNA candidates, particularly targeting highly conserved regions, called
CAPSID (Convenient Application Program for siRNA Design). Using
CAPSID, user can easily extract conserved patterns optimized for siRNA
design in multiple mRNA targets and filter them for the selection of highly
potent siRNA candidates by hierarchical selection criteria. This software
enabled us to readily design a siRNA that targets a conserved
multi-enteroviral region within the virus genomes. Using in vivo assays, we
also found that these siRNAs exhibited potent long-lasting antiviral effects
against related enteroviruses. These findings indicate that CAPSID can be
used to design effective siRNAs from highly conserved sequence patterns and
may contribute to the development of siRNA-based antiviral therapeutic agents
against highly divergent viral genomes. Thus, this work may have led to
optimism through the possibility that siRNA might become a valuable tool for
fighting enterovirus-related human diseases.

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S4-3

Nanomaterials for oligonucleotide based drug delivery

Dal-Hee Min

Department of Chemistry and KI for the BioCentury, KAIST,

Daejeon, 305-701, Republic of Korea

Biotechnology is an emerging area of scientific and technological

opportunity, which requires multi-disciplinary efforts ranging from basic
sciences to engineering and technological development. A major theme of
biotechnology includes development of micro/nanoscale tools for studying
biosystems. Specific research fields include developing platforms to assay
biological and biochemical events—enzymatic activity assays and biomolecular
interactions—for diagnostic tools and/or drug screening. In another approach,
nanoparticles such as qdots are actively harnessed for biosensors, cancer
imaging, and targeted drug delivery in the same realm. The talk will cover
applications of micro/nano-biotechnology in biomedical research area,
emphasizing development of multi-functional nanoparticlesfor oligonucleotide
(siRNA, ASDNA, etc.) based drug delivery.

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S4-4

Toward the development of RNA-based therapeutics:

siRNAs and aptamers
Chan Il Chang, Hye Suk Kang, Jae Wook Yoo, Sun Woo Hong, Pooja Dua,
Shi Eun Lee, and Dong-ki Lee

Global Research Laboratory for RNAi Medicine, Department of Chemistry,

Sungkyunkwan University, Suwon, Republic of Korea

1. Specific knock-down of cellular gene expression in mammalian cells can

be readily achieved using small interfering RNAs (siRNAs). The high
efficiency and specificity of gene silencing by siRNAs makes it a powerful
tool for functional genomics and promising future therapeutics. The structure
of siRNA utilized by most researchers is 19bp duplex with 2 nt 3’-overhang.
Here, we show that a novel asymmetric shorter-duplex siRNA structure
(termed asiRNA) can effectively knock down gene expression in a human
cell line, with reduced non-specific effects. We have also found that, contrary
to the previous belief, dsRNAs longer than 30 bp can trigger RNAi without
non-specific antiviral responses in mammalian cells. Based on this, we
designed novel siRNA structural variants which can simultaneously target
multiple genes. These multi-target siRNAs (msiRNAs) execute efficient
knock-down of multiple target genes as a single molecular entity. Overall,
our findings expand the structural diversity of siRNAs suitable for cellular
gene silencing.

2. Aptamers are short, single-stranded oligonucleotides which, like

antibodies, can fold into specific three-dimensional structures to recognize
target molecules such as small chemicals, proteins, or even cells. We have
been using cell SELEX technology to isolate aptamers which can either
discriminate different cell types, or specifically recognize cells which express
membrane receptor proteins. Using this strategy, we isolated RNA aptamers
which specifically recognize breast- and pancreatic cancer cells. We discuss
the potential application of these aptamers as cancer type-specific therapeutics,
and drug delivery vehicles.

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S4-5

Oligonucleotides for therapeutic application in vitro:

RNA-cleaving oligodeoxyribozyme and inhibitory RNA aptamer
Dong-Eun Kim
Department of Bioscience and Biotechnology, Konkuk University,
Seoul 143-701, Republic of Korea

Nucleic acids provide an enormous potential as a therapeutic reagent against

viral diseases and cancers. Here I show attempts to develop two types of
therapeutic nucleic acids targeting leukemia, viral disease and colon cancer
oligodeoxyribozyme and RNA aptamer.
A class of antisense oligodeoxyribozymes, known as the 10-23 DNAzyme,
has been shown to efficiently cleave target RNA at purine-pyrimidine
junctions in vitro. A screening strategy was performed to identify accessible
cleavage sites for DNAzyme in the target RNA, Hepatitis C virus
nonstructural gene 3 (HCV NS3) that encodes viral helicase and protease,
starting from a pool of random DNAzyme library. The screening procedure
identified 18 potential cleavage sites in the target RNA. Corresponding
DNAzymes were constructed for the selected target sites and were tested for
RNA-cleavage in vitro. The selected DNAzymes, when transfected to the
human hepatoma cells harboring the HCV subgenomic replicon RNA,
efficiently inhibited HCV RNA replication in cells by reducing expression of
HCV NS3 RNA and protein. Thus, the selected oligonucleotides as well as
the selection strategy can be applicable for a new class of anti-HCV drugs
asantisense oligonucleotides-based therapy. The same approach with the
DNAzyme screening has been executed to inhibit b-catenin expression in the
colon cancer cells. Abnormality in Wnt/beta-catenin signaling causes colorectal
cancer, which has been observed in large portion of patients with mutation in
b-catenin and APC. DNAzymes have been selected to recognize and cleave
the b-catenin RNA by the library screening method, and the selected
DNAzymes have shown effective degradation of beta-catenin RNA and
subsequent expression of the gene.
Severe Acute Respiratory Syndrome (SARS) that caused almost 800 victims
requires a development of efficient inhibitor against SARS coronavirus (SCV).
RNA aptamers have been isolated against SCV NTPase/Helicase from RNA
library containing random sequences of 40 nt using in vitro selection
technique (SELEX). The isolated RNAs were observed to efficiently inhibit
double-stranded DNA unwinding activity of the helicase by up to ~85 % with
an IC50 value of 1.2 nM. These results suggest that the pool of selected
aptamers might be potentially useful as anti-SCV reagents.

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- 32 -