RNF19 gene located on human chromosome 8q22.2 has showed 4.4 kb transcript and expressed ubiquitously in various tissues. RNF19 gene coding dorfin protein that carry out E3 ubiquitin ligase, and highly conserved in pig, dog and cattle. RNF19 gene containing MaLR (mammalian LTR-retrotransposon) element in the first intron. Here we found its alternatively spliced transcript variants which derived from MaLR insertion. The MaLR-derived promoter transcripts are detected as two different types in all tissues examined, while breast tissue only showed three variant types. Reporter gene assay of the promoter activity of MaLR element on RNF19 gene indicated good activity in human colon carcinoma cells (HCT-116). These findings suggest that the MaLR element acquired the role of transcriptional regulation of RNF19 gene in various human tissues during primate evolution. .

- cloned from the anterior horn tissues

분류 연구

genome

transcriptome

proteome

genome 수준에서의 연구는이루어지지 않았음

cDNA 서열결정northern blot 으로 발현패턴 확인ALS 에서의 전사체 발현양 연구

E3 activity 의 확인dorfin 이 존재하는 위치확인신경퇴행성질병과의 관련성 연구RBRfamily 의 계통연구parkin 과의 homology 연구활성메카니즘에 관련된 연구 (VCP 관련 )CaR 의 degradation 에 미치는 영향 연구

NM_015435.3 

Parkinson’s disease Amyotrophic lateral sclerosis

Other region16%

Gene-related Sequence

36%

LINE20%

SINE13%

Coding sequence3%

Pseudogene1%

HERV element8%DNA element

3%

RT-PCR

RealtimeRT-PCR Cloning &

Sequencing

Promoterassay

Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006.Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells.Arch. Virol. [Epub ahead of print]

Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003.Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and amyotrophic lateral sclerosis.Am J Pathol. 163(2):609-619.

Mar

ker

Adr

enal

gla

nd

Adu

lt br

ain

Fet

al b

rain

Fet

al li

ver

Hea

rtK

idne

yL

iver

Lun

g

Bon

e m

arro

wC

ereb

ellu

m

Skel

etal

mus

cle

Mar

ker

Pla

cent

a

Spin

al c

ord

Tes

tisT

hym

usT

hyro

id

Tra

chea

Ute

rus

Pro

stat

e

Dorfin

Gapdh

494 bp

195 bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Bre

ast

(N)

Bre

ast

(C)

Mar

ker

AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC

R LE GN G DV IG A

R LE GN G DV IG A IS

GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC

CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG

CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG

TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT

TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT

TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA

GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA

CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG

CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA

ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC

AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC

AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA

GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC

CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG

CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG

TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT

TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT

TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA

GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA

CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG

CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA

ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC

Primer S2

Primer AS2

M Q E Q E I G F S KI Y

G

N E

CL NV T PD SV I TL IS L D

M LS RH Q GM DS R LD SQ S

A SS SV L SP KV K PA KK R R

R SI GI S FL KR K ND RK K S

IS SE I

M LS RH Q GM DS R LD SQ S

A SS SV L SP KV K PA KK R R

R SI GI S FL KR K ND RK K S

SE I

AluJ element

AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA

556 bp430 bp315 bp

(A) (B)

(C)

3 111 2

MaLR AluJ

AluJMaLR

2 101

AluJMaLR

2 111 3

126 bp 115 bp

1)

2)

3)

123

0

0.01

0.02

0.03

0.04

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

MaLR-derived promoter transcript (556 bp)

MaLR-derived promoter transcript (430 bp)

Rel

ativ

e E

xpre

ssio

n

Bone marrow

Cerebellum Kidney

Lung

Testis

Reverse

Forward

Reverse

Forward

Control

Relative Luciferase Activity

(Fold of pGL-2 control)

0 2 4 6 8 10 12 14 16 18 20 22

Cos7

HCT116

CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA GGGG

TTTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA GGTAGCCT

CA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG AAGACGACGC

GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG TAACCGCAAG CC

GAGGAATG CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT CTCCCCTCAA GCCCC

AGTGG GAGCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA ACTGTAAGAG AAATGTCCA

T TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG CCCCAGGAAA CTACTA

.

GATA-1

GATA-2

Nkx2-5 Nkx2-5

Whn ELF-1

+1 TRANSCRIPTION START SITE

In silico analysis of transcription binding sites of MaLR element

Human

Dog

Pig

Cattle

Mouse

Rat

Chicken

Zebrafish

100

100

100

42

77

0.05

100

77

42

100

100

100

Human

Dog

Pig

Cattle

Mouse

Rat

ZebrafishChicken

35cycle

94 ℃ : 40sec

56 ℃ : 30sec

72 ℃ : 40sec

40cycle(Sybr green)94 ℃ : 10sec

56 ℃ : 15sec

72 ℃ : 15sec

QiaquickGel-extraction

PromegaT-easyVector

EcoR-I(DH5α)

Modified PGL2-vector

HCT116Cos7

Lipofectamine

Dual luciferaseAssay