―Review―

Basic and Clinical Studies on Functional RNA Molecules
for Advanced Medical Technologies
Toshihiro Takizawa1, Akihiko Gemma2, Kumiko Ui-Tei3, Yasunori Aizawa4,
Yoel Sadovsky5, John M. Robinson6, Masahiro Seike2 and Koichi Miyake7
1

Division of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School

2

Division of Pulmonary Medicine, Infection Diseases and Oncology, Graduate School of Medicine, Nippon Medical School
3

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo
4

Center for Biological Resources and Informatics, Tokyo Institute of Technology
5Magee-Womens Research Institute, University of Pittsburgh, USA

6

Department of Physiology and Cell Biology, Ohio State University, USA

7

Division of Biochemistry and Molecular Biology, Graduate School of Medicine, and Division of Gene Therapy Research Center for
Advanced Medical Technology, Nippon Medical School

Abstract
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are functional RNA molecules
that

have

recently

emergedasimportantregulators

of

gene

expression

at

the

posttranscriptional or translational level. The RNA interference effects of siRNA on gene
expression make it a valuable research tool for knocking down the expression of genes inmammalian cells in vitro and in vivo enabling the elucidation of molecular mechanisms
underlying human diseases. Endogenous miRNAs are involved in a variety of physiological andpathological processes in humans. In this mini-review we first address the synthesis,
mechanisms of action, and functions of siRNAs. Then, we focus on recent advances andtechnologies in miRNA and protein research of the human placenta. Next, we discuss theclinical applications of miRNA in lung cancer. We also touch on “long” noncoding RNAs fromintergenic regions of the human genome. This review article is based on a presentation given
at a symposium entitled Basic and Clinical Studies on Functional RNA Molecules for Advanced
Medical Technologies held at Nippon Medical School in Tokyo, Japan, on November 7, 2009.
(J Nippon Med Sch 2010; 77: 71―79)
Key words: small interfering RNA, microRNA, noncoding RNA, placenta, lung cancer

Medical Technologies held at Nippon Medical School in
Introduction

Tokyo, Japan, on November 7, 2009. The aim of thepresentation was to describe recent advances inThis review article is based on a presentation

medical technology related tofunctionalRNAgiven at a symposium entitled Basic and Clinicalmolecules and to show how RNA molecules canStudies on Functional RNA Molecules for Advanced

improve our understanding of pathophysiological

Correspondence to Toshihiro Takizawa, Division of Molecular Medicine and Anatomy, Graduate School of Medicine,
Nippon Medical School, 1―1―5 Sendagi, Bunkyo-ku, Tokyo 113―8602, Japan
E-mail: t-takizawa@nms.ac.jp
Journal Website (http:!
!
www.nms.ac.jp!
jnms!
)
J Nippon Med Sch 2010; 77 (2)71T. Takizawa, et alfunctions related to human health and disease.

Thus

Kumiko Ui-Tei presented effective and target-

relationship between siRNA sequence and RNAi

specific

effectgenesilencing,regulated

by

small

Ui-Teiinandcolleagues

mammaliancells 4.

investigated
They

the

presented

interfering RNA (siRNA) sequences. Koichi Miyake

principles that govern siRNA sequence preference,

demonstrated application of viral vector mediatedby which highly effective siRNAs can be readilysiRNA expression for creating disease models anddesigned. The rules indicate that siRNAs thattreatment. John M. Robinson showed placental

simultaneously satisfy all 4 of the following sequence

proteomics

and

short

(shRNA)

conditions are capable of inducing highly effective

technology

forfunctional

proteins

gene silencing in mammalian cells: (i) A!
U at the 5́

identified in human placenta. Yoel Sadovsky gave a

end of the guide strand; (ii) G!
C at the 5́ end of thelectureplacental

passenger strand; (iii) at least 5 A!
U residues in themacroRNA and microRNA (miRNA) expression.

5́ terminal one-third of the guide strand; and (iv) the

Masahiro Seike revealed miRNA signatures in lungabsence of any GC stretch of more than 9cancer. Finally, Yasunori Aizawa presented “long”

nucleotides in length. For 97.7% of mRNA sequences

noncoding RNAs (ncRNAs) from intergenic regions

registered in the National Center for Biotechnologyof the human genome. The studies discussed here

Information Reference Sequence (RefSeq) database,provide new insights into basic and clinical medicine.

at least 1 effective siRNA sequence has beenon

integrated

hairpinRNA

analysisofanalysisofdesigned5. Essentially the same rules for siRNA
Effective and Target-specific Gene Silencing

sequence preference were found applicable to DNAbased RNAi in mammalian cells.

Regulated by SiRNA Sequences

growingbodyofevidence

from

However,alarge-scale

Small regulatory RNAs, including siRNAs andknockdown experiments suggests that siRNA could

miRNAs, are crucial regulators of gene expression at

generate off-target effects through a mechanism

silencingsimilar to that of target silencing by miRNAs6,7. The

encompasses a group of mechanistically related3́ untranslated regions (3́ UTRs) of off-target

pathways that produce small ncRNA molecules,transcripts or miRNA targets are complementary towhich modulate protein expression via mRNA

the seed region of the guide strand, nucleotide

degradation or translational repression. In RNA

positions 2 to 8 measured from the 5́ end. Withininterference (RNAi), long double-stranded RNAs arecells, the siRNA guide strand binds to Ago to form

processed by the cytoplasmic RNase III enzyme

RISC. In RISC, the seed nucleotides are presumed to

Dicer into 21- to 23-nucleotide siRNAs. The siRNAbe present on the surface of Ago in a quasihelicalguide strand that is incorporated into the RNA-

form to serve as the entry or nucleation site forinduced silencing complex (RISC) recognizes closely

mRNA8. However, little was known about theor completely matched target sequences, and a coremolecular basis that determines the efficiency ofcomponent of RISC, the Argonaute (Ago) protein,seed-dependent

mediates the sequence-specific cleavage of target

miRNA. To clarify this point, Ui-Tei and colleagues

theposttranscriptional

1,2level .

RNA

3

mRNAs .

gene

silencing

by

siRNA

and

examined the relationship between the stability of

The siRNA-based RNAi has been used as a

the seed-target duplex and the capability of siRNA

research tool to control the expression of specificto induce off-target effects9. They found that thegenes in numerous experimental organisms and has

capability of siRNA to induce off-target effects

potential as a therapeutic strategy for several

correlated strongly with the calculated melting

human diseases. However, it has not been usable fortemperature or standard free energy change for

the

for

formation of the protein-free seed-target duplex. The

mammalian functional genomics, because only athermodynamic stabilities of the duplexes werelimited fraction of siRNAs appearof

calculated from the seed sequences, indicating that

producing highly effective RNAi in mammalian cells.

the seed sequence is the major factor in determining

72

large-scalegenesilencingessential
capableJ Nippon Med Sch 2010; 77 (2)Functional RNA Molecules for Medicine

Fi
g.1 Dr
ugi
nduc
i
bl
ee
xpr
e
s
s
i
o
ns
ys
t
e
mo
fs
i
RNA us
i
ngl
e
nt
i
vi
r
a
lve
c
t
o
r
s
.
db
o
x
,
KRAB;
Se
l
f
i
na
c
t
i
v
a
t
i
ngLTR,
SI
N.
Abbr
e
vi
a
t
i
o
ns
:
Kr
up
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s
s
o
c
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a
t
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the degree of off-target gene silencing. Furthermore,

matches10. Thus, their procedure to select siRNA

unlike RNAi, off-target effects were completely

should be applied to diverse mammalian RNAi

abolished by introduction of a G : U wobble pair intoexperiments including functional genomics.

the seed-target duplex, indicating that Watson-Crickpairing in the seed-target duplex is also essential for

Application of Viral Vector Mediated SiRNA

off-target gene silencing. In this case, off-target

Expression for Creating Disease

effects are likely avoided because of the structural

Models and Treatment

perturbation of the seed-target duplex on thesurface of Ago protein but not the duplex stability.

SiRNAs have been shown to inhibit endogenous

Unlike the off-target gene silencing, intended gene

genes in a sequence-specific manner in mammaliansilencing by RNAi was significantly tolerant to thecells11,12. Posttranscriptional gene silencing by siRNAspresence of a G : U pair in the seed region. Ui-Tei

has quickly emerged as a powerful tool for genetic

and colleagues, therefore, consider that for RNAi,

analysis in mammalian cells and has the potential towhich requires a near-perfect match between target

produce novel therapeutics. Miyake and colleagues

mRNA and the siRNA guide strand, the firsthave developed a novel regulatable expression

recognition of mRNA by the seed region in thesystem of siRNA using lentiviral vectors that canguide strand and the subsequent target recognition

stably transduce both dividing and nondividing

by the nonseed region of the guide strand are highly

target cells13. This system can achieve the highly

cooperative, and, therefore, the second proof-reading

efficient, drug-inducible knockdown of cellular genes

process can easily surmount the G : U pairing hurdle(Fig. 1). Using this lentiviral-mediated drug-inducible

in the initial mRNA nucleation process.

system, Miyake et al.14 and Flygare et al.15 have

Ui-Tei and colleagues presented that effective andestablished cell-line models for ribosomal protein S19

target-specific gene silencing is closely related to the(RPS19)-deficient Diamond-Blackfan anemia (DBA),

siRNA sequence, and it was found that >94% of

as no models exist for RPS19-deficient DBA, and thehuman mRNAs in the RefSeq database possess atmolecular pathogenesis is unknown. To establish an

least 1 target sequence capable of producing: 1)

in vitro inducible model for DBA, human erythroid

functional siRNAs that satisfy these guidelines, 2)

leukemic cell lines TF-1 and UT-7 cells weresiRNAs with reduced seed-dependent off-target

cotransduced with a lentiviral vector expressing theeffects by considering the thermodynamic stability

green fluorescent protein (GFP) gene and an siRNAof the seed-target duplex, and 3) siRNAs that do notagainsthit

regulatoryanynontargeted

genes

J Nippon Med Sch 2010; 77 (2)withnear-perfect

RPS19

controlledelement

and

by

a

anothertet

operatortransactivator73

T. Takizawa, et al

vector containing the red fluorescent protein (RFP)eye, and muscle was observed in AAV-9, AAV-8, andgenetetracycline-

AAV-8 injected mice respectively. Because the AAV

and

a

cDNA

encodinga

Following

vector is nonpathogenic and can transduce withtransduction, the RFP-positive and GFP-negative cellhigh efficiency in a wide variety of organs in vivo,

population was isolated with flow cytometry. Upon

the appropriate AAV vector serotype is a powerful

incubation with doxycycline, more than 98% of cells

tool for the delivery in vivo of siRNA and!
or miRNA.

controllable

expressedtranscriptional

GFP

and

repressor.

thesiRNA.

Significant

suppression of erythroid differentiation, cell growth,Placental Proteomics: a Gateway toand colony formation was observed in cells treated

Functional Analysis

with the siRNA against RPS19 but not in cells
treated with a control vector. These novel cell lines

Patterns of protein expression in tissues, cells, orrepresent models for RPS19-deficient DBA and cansubcellular components are characteristic of specificbe used to identify the molecular mechanisms inphysiological,

RPS19-deficient DBA16. Thus, this novel regulatableconditions. Therefore, the understanding of protein

expression system of siRNA using lentiviral vector isexpression profiles of a sample under specificuseful for the knockdown of cellular genes.

conditions is highly desirable. Proteomics refers to

Another potential viral vector to express siRNA is
17,18

adeno-associated viral (AAV) vector. Systemic

developmental,orpathologicalthe identification and characterization of protein
components in a biological sample. Ideally, thegene delivery by AAV vectors has been successfully

proteins

used for long-term expression of transgenes to treat

posttranslational state determined. While the human

genetic diseases and cancer. Recently, a number ofgenome has about 25,000 genes, the repertoire ofnovel AAV serotypes have been isolated fromprotein expression in humans may be closer to 1

nonhuman primates. To analyze the utility of AAV

million21. This dramatic amplification of the protein

vectors in vivo, Miyake and colleagues examined therepertoire is due to alternative splicing of genes andexpression patterns and time course of expressions

posttranslational modifications to which proteins are19

for different serotypes of AAV after the injection of
the vectors into mice. They injected AAV vectors
11

would

be

quantified,

and

their

subject.
A detailed analysis of protein expression invectorhealthy placentas is essential because the protein

genome each) encoding luciferase gene into the tailrepertoire of the placenta is not known. Thesevain

real-timebaseline data are essential for studies comparing

monitoring of transgenes with a bioluminescent innormal and diseased placentas. However, global(types 1, 2, 4, 5, 6, 7, 8, 9, and 10: 1
of

DDY

mice

and

10

performed

luciferase

proteomics analysis of a complex structure, such as

expression level in the whole body was observed in

the placenta, is not feasible currently22. The best

mice 1 week after receiving injections of AAV-8 (8>available strategy to deal with this problem is to9>7>10>6>5>1>4>2). The vectors AAV-1, -6, -7, -8,

simplify the placenta by fractionation; that is, to-9, and -10 induced rapid expression and reach aanalyze subproteomes. Robinson and colleagues haveplateau at 4 weeks, whereas vectors AAV-2, -4, andfocused-5 were slower to induce expression of the reporter

subproteome, the apical plasma membrane of thegene. Expression was prominent in the liver, muscle,

syncytiotrophoblast (STB) of the human placenta.

and heart in mice receiving injections of vectors

Specifically, they have focused on the membrane

AAV-1, -2, -5, -6, -7, -8, -9, -10, and in the lung of miceproteins in that subproteome. Defining this proteome

receivingis desirable considering the importance of thisvivo

imagingsystem.

injections

of

The

highest

AAV-4.

Miyakeandcolleagues also analyzed the transduction efficiency

on

the

maternal-placental

interfacemembrane to placenta function.

of several AAV vectors expressing luciferase and!
or

Enrichment of the apical plasma membrane of the

GFP gene by direct injection into brain, eye, andSTB to the greatest extent possible prior to themuscle20. The highest expression level in the brain,

proteomics analysis is important. The need for74

J Nippon Med Sch 2010; 77 (2)Functional RNA Molecules for Medicine

enrichment is due largely to the dynamic range ofmuscle. Mutations of dysferlin lead to limb girdle

protein expression in biological samples. In serum,

muscular dystrophy and Myoshiʼs myopathy26,27.

the dynamic range can vary over 12 orders ofMoreover, experimental evidence indicates thatmagnitude; in cells, the range can be over 8 orders

dysferlin can serve to repair damaged plasmaof magnitude. This presents a practical problem for

membranes in skeletal muscle28,29. The functions ofidentifying low-abundance proteins23. Robinson anddysferlin and myoferlin in the STB are not known.

colleagues

aRobinson and colleagues have hypothesized thatmodification of one used for the proteomic analysis

dysferlin and, perhaps, myoferlin serve a similar roleof the luminal plasma membrane of rat lungin STB repair. This hypothesis is not easily

have

used

a

procedure

that

is

24

endothelium . In this procedure, the apical plasma

addressed experimentally in the human placenta.

membrane of the STB is coated with cationic

They have turned to primary cytotrophoblasts

colloidal silica, which alters the buoyant density of

isolated from term placenta and cell lines, such as

this membrane relative to the other membranes in

BeWo, to address this question. They have foundthe placenta. Following tissue homogenization, thethat in cytotrophoblasts and BeWo cells dysferlin isapical membrane (actually microvilli) is isolated byexpressed

centrifugation. A detailed account of this methodmononuclear cells. This is similar to the placenta,

25

was recently published . Using this procedure,

where

after

the

cell-cell

STB

fusionexpresses

dysferlin

30,31

in

but

the

cytotrophoblast

placental alkaline phosphatase, the classic marker fortechnology, Robinson and colleagues have generated

the apical plasma membrane of the STB, by 200 tolines of BeWo400

isolated

myoferlin, or lacking both proteins. Robinson andmicrovilli to a series of extraction steps designed tocolleagues are now studying these cell lines todissociate noncovalent protein-protein interactions todetermine the roles of dysferlin and myoferlin in

further enrich for membrane proteins. Incubating

trophoblast biology.

They

then

subjected

the

cells

not

not

Robinson and colleagues were able to enrich fortimes.

doesbutlacking

.

Using

shRNA

dysferlin,lacking

the sample in environments of low salt, high salt,high pH, and urea resulted in a further 5-foldenrichment.

There

was

1,000-

toIntegrated Analysis of Placental MacroRNA

2,000-foldand MiRNA Expressionenrichment in the final sample. No other tissue
plasma membrane has been enriched to this degree.

Intact function of the placenta is essential for theThis material was used for proteomics analysis by

growth and development of the embryo. Within thetandem mass spectrometry.

placenta,

theMore than 500 proteins were identified in theexchange,

nutrition,enriched apical plasma membrane of the STB. These

function,andproteins were characterized in a number of ways.

developing fetus. Sadovskyʼs research goal is to

Robinson and colleagues were particularly interested

understandin those identified proteins not previously known todifferentiation during the latter half of pregnancy

be in the placenta. They have validated theand the response of human trophoblast to injury.

presence of a number of these proteins usingThe

immunoblot analysis. It is important to confirm thedifferentiation and placental dysfunction are far

mass spectrometry data using independent methods,

reaching, typically leading to fetal growth restriction

such as immunoblotting.

or even fetal death, and frequently a need forthetrophoblast

layer

waste

removal,immunologicalprocess

consequencesofgovernsof

supporthuman

abnormalgas

endocrineforthetrophoblast

trophoblast

Robinson and colleagues identified 68 proteins

medically indicated preterm delivery designed toheretofore unknown in the human placenta. Their

prevent further damage32. Newborns surviving theseexperimental attention has focused on 2 of these,

insults

dysferlin and myoferlin. Before their study, dysferlin

including neuro-developmental dysfunction and a

and myoferlin were known to be only in skeletal

greater incidence of the adult metabolic syndrome

J Nippon Med Sch 2010; 77 (2)

are

at

risk

for

lifelong

complications,75

T. Takizawa, et al(including type 2 diabetes, hyperlipidemia, obesity,
33

the impact of hypoxia on trophoblast miRNAs and toand related illnesses) . Whereas the association ofassessthese conditions with placental dysfunction has been

microarrays they defined a set of miRNAs (such as

substantiated,

underlying

miR-205) that exhibits altered expression in hypoxic

trophoblast differentiation and adaptation to injury

human trophoblasts. To define the targets of theseare poorly understood. Furthermore, there is no

miRNA they combined high-throughput profiles ofeffective treatment for this condition. The only

miRNAs and mRNAs from hypoxic trophoblastsintervention to prevent further fetal damage iswith in silico prediction of miRNA targets. Using thisdelivery,computational approach they identified candidate

the

which

mechanismscommonly

leads

to

iatrogenic

theirmolecular

targets.

Using

miRNA

trophoblast target mRNAs to their hypoxia-inducedprematurity.
establish

mRNAs. They further confirmed miRNA targets

implantation and early placental development. Laterusing miRNA reporter technology and mutagenesis

in pregnancy, a different set of signals determinesof specific miRNA binding elements41. Sadovsky andthe differentiation and function of the establishedcolleagues also assayed the level of these miRNAs inplacenta. Exposure of the placenta to insults, such as

the plasma of women with pregnancy complicatedhypoperfusion and cellular hypoxia, adversely affects

by fetal growth restriction.

Diversepathwayscoalescetotrophoblast function and leads to placental injury.
Hypoxia regulates trophoblast gene expression and

MiRNA Signatures in Lung Cancer

adversely affects cell survival, differentiation, andmetabolicfunction,theLung cancer is the leading cause of cancer death

pathophysiology of placental dysfunction and fetal

in Japan and worldwide. The identification of

34,35

growth restriction

which

contributetosensitive and specific biomarkers for prognosis and.

The noncoding, 20- to 24-nucleotide-long miRNAs
are abundant in all metazoan eukaryotes, where

drug sensitivity may have a significant impact onlung cancer treatment strategies.

gene

MiRNAs are small noncoding RNA molecules. These small RNAs are processed fromcomprising 18 to 25 nucleotides which are frequently

longer transcripts carrying a characteristic hairpin

located at previously reported regions for genetic

secondary structure. Currently, the number ofalterations in cancers, suggesting that miRNAs are avalidated mature human miRNAs approaches 1

new class of genes involved in human tumorigenesis.

thousand,of

Recently, miRNAs have also been demonstrated torepressing multiple genes. Not surprisingly, miRNAs

be diagnostic and prognostic biomarkers, including a

have emerged as important regulators of virtuallyrole in monitoring the response to therapy in lungevery biological process associated with tissue

cancer42,43. For example, high miR-155 expression anddevelopment, differentiation, cellular proliferation,

low let7a expression, as independent risk factors,

cell-type

homeostasis.

have a negative prognostic impact in patients with

Consequently, dysregulation of miRNAs has beenlung adenocarcinoma42. Inhibition of the miR-21 byimplicatedregulatory

enhanced epidermal growth factor receptor tyrosine

networks. Indeed, several pathologies have beenkinase inhibitor (EGFR-TKI)-induced apoptosis in

they

act

posttranscriptionally
36,37

expression

withindividual

specific
inthefunction,tosilencemiRNAsanddysfunction

capableof
38,39

linked to altered expression of miRNA

.

lung

cancercells,

which

showedintermediate

Hypoxia was recently shown to impact miRNAsensitivity to the EGFR-TKI43. In miRNA-drug

expression in diverse cellular contexts. Sadovsky

correlation analyses, Seike and colleagues haveand colleagues have previously shown that theidentified unique miRNA expression patterns thatmiRNA biosynthetic pathway is functional in humancorrelate with the drug sensitivity to each oftrophoblasts exposed to hypoxic stress and that thecommonly used 10 anticancer agents. These findings

expression of key miRNA machinery proteins is notsuggest that miRNAs may serve as novel diagnostic

altered by hypoxia40. They now seek to understandmarkers and therapeutic targets in lung cancer.

76

J Nippon Med Sch 2010; 77 (2)Functional RNA Molecules for Medicine

genes and signaling pathways52,53. Thus, Aizawa andcolleagues

NcRNAs from Intergenic Regions of the

expected

that

some

TUFs

were

composed of signaling networks for stem cell

Human Genome

differentiation

anddynamicallychangedRecent mammalian transcriptome analyses havetranscription activities while differentiation proceeds.

44―46

.

On the basis of this speculation, they designed a

These transcripts differ in size, spliced mode,

DNA microarray for approximately 5,500 humanpolyadenylation, status and intracellular location.

TUFs and monitored the transcription levels for the

Because their functional roles are currently unclear,first 7 days of hMSC adipogenesis and osteogenesis54.

they are designated as transcripts of unknown

Consequently, Aizawa and colleagues obtained 6

uncovered tens of thousands of novel transcripts

47,48

function (TUFs)
functional. In the postgenomic era, thesignificance

of

TUFs

has

received

strongcandidatesoffunctional

TUFs,

which

exhibited marked and lineage-specific changes inabundance.

considerable attention.
A common feature of TUFs is low coding

Subsequent sequence analysis of the resultant

potential. In sharp contrast with mRNAs, which

TUFs allowed us to unexpectedly find 1 of the 6

generally contain a single large open reading frame

TUFs, named adipogenesis downregulating gene 3(ORF), TUFs have many start and stop codons(AGD3). Among the 6 TUFs, AGD3 is the only one

scatterednucleotidethat has been reported in the literature and wassequences. Thus, TUFs always have several small

found to be highly expressed in colon cancer as a

ORFs and yet rarely resemble any protein families.

ncRNA 55.

Owing to these features, it is widely believed thattranscripts obtained from the human AGD3 syntenic

most TUFs are “long” ncRNAs. For more than aregions in thedecade, long and polyadenylated ncRNA genes, suchgenomes, Aizawa and colleagues demonstrated that

as XIST (X (inactive)-specific transcript), have been

all these AGD3 orthologs contain small sequence-

known to be functionally essential to gene regulation

homologous ORFs. They then prepared polyclonalthroughout

theentire49

However,bymouse,

examiningchicken,orthologous

andzebrafishon chromosomal and global scales . Recently, theantibody

number of functional ncRNAs has been expanded

confirmed with Western blotting that the protein

AGD 3-derived

peptides

and

This

was indeed produced in hMSCs54. To our knowledge,

growing list of functional long ncRNAs does not,

AGD3 is the first mammalian TUF that washowever, exclude the possibility that any TUFs

experimentally

encode small proteins. Indeed, it was found that

subsequent pathway analysis using protein kinase A

novel and small proteins were encoded in transcripts( PKA )

that were originally believed to be ncRNAs inmethylxanthine and forskolin) showed that AGD3

invertebrates.

using

different

experimentalbetranslated.

compoundsThe( 3-isobutyl-1-

by the PKA pathway. With the observed up-

RNA and!
or protein genes.

regulation in colon tumors, this finding may suggest

overview

ofexpectinhibitor

toexpression is suppressed during hMSC adipogenesis

an

can

shown

that

gain

one

approaches .

mammalian TUFs include unprecedented types ofTo

Therefore,

50

againstbiologically

activemammalian TUFs, Aizawa and colleagues screenedforcandidatesoffunctional

TUFs

and

that AGD3 gene is invovled in cell proliferation.
Serendipitous finding of AGD3 brought up athenfascinating hypothesis: TUFs could be a valuable

investigated sequences of the identified TUFs. This

reservoir of new protein genes. As mentioned above,

study focused on human TUFs that are involved in

ORFs on TUFs are all small (<100 amino acids), andmultilineage differentiation of human mesenchymal

the amino acid sequences are rarely categorized into

stem cells (hMSCs). In general, stem cells are muchany

more transcriptionally active than are differentiatedoriginated protein genes would be able to create

51

known

proteinfamilies.

Therefore,

TUF-

cells , and induction of differentiation triggers global

new protein families. Indeed, on the basis of theand stepwise activation and!
or inactivation of many

sequence features of the AGD3 transcription unit,

J Nippon Med Sch 2010; 77 (2)77T. Takizawa, et al

Aizawa and colleagues carried out a pilot screeningof human TUFs for new protein genes anddiscovered several protein gene candidates, which
all have orthologs in mammals or vertebrates but nosequence

similarities

with

any

known

proteinfamilies. Future studies may demonstrate as-yetunidentified molecular mechanisms that these newproteins govern in cells.
Future Directions
The technological advances and applications offunctional RNA molecules for medicine discussed in
the symposium provide important insights intomolecular mechanisms affecting human health anddisease and should eventually lead to the discovery
of diagnostic biomarkers and the development ofnovel gene therapies.
Acknowledgement: We thank Dr. Tetsuo Ikezono, Dr.
Atsushi Watanabe, Dr. Osamu Ishibashi, and Mr. Takuji
Kosuge for their support and assistance. This work was
supported in part by the “Research Core” Project forPrivate University: Matching Fund Subsidy from the
Ministry of Education, Culture, Sports, Science andTechnology, Japan.

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(Received, February 9, 2010)(Accepted,

March 2, 2010)79