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제 4 회 부산대학교 생물학과 학술제. Mobile Genetic Element. Nonviral Retroposon LINE Element. 지도 교수 : 김 희 수 교수님 발 표 자 : 문 요 섭 참 가 자 : 김 영 균 , 윤 성 용. Retrovirus (HIV). Receptor binding proteins. Lipid envelope. RNase. Viral RNA. Capsid : core proteins. Major Groups of Retroviruses.

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slide4

제 4 회 부산대학교 생물학과 학술제

Mobile Genetic Element

Nonviral Retroposon LINE Element

지도 교수 : 김 희 수 교수님

발 표 자 : 문 요 섭

참 가 자 : 김 영 균, 윤 성 용

slide5

Retrovirus (HIV)

Receptor

binding proteins

Lipid envelope

RNase

Viral RNA

Capsid : core proteins

slide7

Genomic Structure of Retrovirus

Coding region

LTR

LTR

Integrated retroviral DNA

U3

R

U5

U3

R

U5

Host-cell DNA

Poly(A) site

Start site

RNA polymerase II

Primary transcript

5`

3`

RNA-processing enzymes

Poly(A) polymerase

Retroviral RNA genome

(A)n

slide8

Generation of LTR

R

U5

tRNA

3`

5`

tRNA

Genimic RNA

(A)n

5`

3`

PBS

R

U5

Coding region

U3

R

DNA

3`

tRNA extended

R

U5

tRNA

to form DNA copy

(A)n

3`

5`

R

U5

PBS

Coding region

U3

R

Hybrid RNA

removed

(A)n

PBS

Coding region

U3

R

3`

R

U5

tRNA

First jump

(A)n

PBS

Coding region

U3

R

3`

tRNA

U3

R

U5

DNA strand extended

from 3` end

PBS

Coding region

U3

R

(A)n

slide9

Must hybrid RNA removed

tRNA

PBS

U3

R

U5

3`

5`

5`

3`

3`

tRNA

U3

R

U5

3` end of second DNA strand

5`

synthesized

3`

PBS

U3

R

U5

5`

3`

U3

R

U5

Remaining hybrid RNA

and tRNA removed

3`

PBS

U3

R

U5

3`

5`

U3

R

U5

Second jump

3`

PBS

5`

U3

R

U5

3`

U3

R

U5

5`

Both strands

completed by

5`

3`

PBS

U3

R

U5

synthesis from

Retroviral DNA

LTR

3` ends

LTR

slide10

Replication Mechanism of Retrovirus

Absorption to Specific Receptor

Endocytosis

Reverse transcription

Provirus DNA

Transport to nucleus

Double strand

of DNA

Translation

Transcription

mRNA

Nuclear membrane

Assembly

Cytoplasm

Release

Budding

slide12

Mobile Elements in Mammalian Genome

D D E

P

ORF1

ORF2

Poly(A)

TTTT

Poly(A)

Class

Example

Structure

Mechanism

Transposase

‘cut’ or ‘copy

and paste’

Transposon

mariner

Autonomous

Retrotransposons

Truncated

HERVs

LTR-containing

Retroviral-like

LTR

ORF1

ORF2

LTR

Target primed reverse

transcription(TPRT)

Non-LTR

L1

EN RT

R-Alu

L-Alu

Non-autonomous

Alu

TPRT?

terminator

retrotransposons

A-rich

linker

Processed

pseudogene

TPRT?

slide13

Mechanism of L1 Retrotransposon

P

ORF1

ORF2

Poly(A)

EN RT

AAAAn

AAAAn

Integration of truncated L1

at new chromosomal site

Cytoplasm

Nucleus

Poly(A)

Target primed reverse

AAAAA

transcription(TPRT)

Transcription processing

3`OH-TTTTT

5`

mRNA export

ORF2

5`

Import or entry

during mitosis

ORF2

5`

AAAAn

Transcription and

ORF2

ORF2

RNP assembly;

p40

Post-translational

5`

modification

p40

slide14

Retroelement

RNA intermediate

- LTR element

+ LTR element

Retroposon

- env

+ env

Retrotransposon

- RT

+ RT

LTR

ORF1

ORF2

LTR

SINE

Yeast Ty1/copia/truncated HERVs

P

Poly(A)

LTR

LTR

Human Alu

Human THE1

Retrovirus

LINE

LTR

gag

pol

env

LTR

P

ORF1

ORF2

Full-length HERVs/exogenous retrovirus

Poly(A)

L1

slide15

Transposon & Retrotransposon

Insertion sequence or transposon

Retrotransposon

Donor DNA

Donor DNA

Flanking DNA

RNA polymerase

RNA intermediate

Reverse transcriptase

DNA intermediates

Target DNA

Transposed mobile element

slide16

Non-LTR Retro-element

LTR

ORF1

ORF2

LTR

P

Poly(A)

LTR

LTR

Human Alu

LTR

gag

pol

env

LTR

P

ORF1

ORF2

Poly(A)

L1

Retroelement

RNA intermediate

- LTR element

+ LTR element

Retroposon

- env

+ env

Retrotransposon

- RT

+ RT

SINE

Yeast Ty1/copia/truncated HERVs

Human THE1

Retrovirus

LINE

slide17

Evolution & Phylogeny of Non-LTR Retrotransposable Element

CLADE

100

CRE

RT

100

R2

RT

98

R4

RT

90

L1

APE

RT

99

APE

RTE

APE

RT

RT

98

APE

RT

Tad

APE

RT

RNH

82

82

99

APE

RT

R1

APE

RT

RNH

100

LOA

APE

RT

RNH

100

76

APE

RT

RNH

I

RNH

APE

RT

RNH

100

Jockey

APE

RT

100

56

CR1

APE

RT

250 aa

slide18

Structure of LINE Element

~

(~1 kb)

ORF1

~

(~4 kb)

ORF2

3`

5`

5`

3`

A/T-rich region

Multiple

stop codons

Target-site direct repeat

Coding region

(~1 kb)

~

slide19

Synthesis of L1 LINE Element

3`

5`

AAAAAAAAA

TTTTTT

TTTTTTTTTT

AAAAAA

5`

3`

L1 element

Flanking DNA

1) An RNA polymerase transcribes bottom strand

AAAAAAAAA

UUUUUU

5`

3`

2) RNA fold back on itself with Us and As hybridizing

3) 3` end primes synthesis of DNA from RNA

template by L1 reverse transcriptase

AAAAAAAAA

5`

3`

TTTUUUUUU

cDNA

4) cDNA serves as template for synthesis of double-stranded L1 DNA

5) Unknown mechanism insert L1 element into cellular DNA

slide20

Retroposon Model of L1 Movement

SDR

Inserted

functional L1

ORF1

ORF2

Transcription

L1 mRNA

A(n)

5`

3`

ORF-2 product

DNA/RNA hybrid

+

Target site

???

Transposed

5` truncated L1

New SDR

slide21

Various Model for L1 Element

(A)n

3`

5`

(T)n

5`

5`

3`

L1 RNA

3`

3`

ORF-2 RTase

(A)n

3`

(A)n

5`

5`

5`

Repair synthesis

cDNA

(T)n

3`

(T)n

5`

RNAase H

3`

(T)n

5`

(A)n

3`

5`

3`

S.S. nuclease

(partial)

Random priming

Integration

(T)n

5`

(T)n

3`

5`

(A)n

3`

(A)n

5`

3`

S.S. nuclease (nick)

Inverted segment

(T)n

3`

5`

5` Inverted/deleted L1

5`

Repair synthesis

S.S. nuclease

(complete)

(A)n

3`

3`

(T)n

3`

(T)n

5`

5`

5`

(A)n

5`

3`

(A)n

3`

Integration

Integration

(T)n

(T)n

3`

5`

3’`

5`

5`

(A)n

5`

(A)n

3`

3`

Inverted segment

5` Truncated L1

5` Inverted L1

slide22

Genomic Diversity by L1 LINE Element

ORF1

ORF2

An

An

C

EN

RT

Transduction

An

An

An

An

An

slide23

Promoter Arrays & L1 Transposon

Parental L1Md element

208 bp repeats

SDR

4 2/3 copies

ORF-2

(A)n

ORF-1

SDR

3 2/3

2 2/3

mRNAs

1 2/3

2/3

Functional progeny L1Md element

+

Non-functional progeny L1Md element

slide24

Comparative Analysis of L1Md & L1Hs

ORF-1

5`UTR

3`UTR

(A)n

ORF-2

L1Md

ORF-2

ORF-1

(A)n

5’UTR

L1Hs

3’UTR

slide25

Common 3` Sequence of LINE & SINE

SINE

tRNA-related region

tRNA-unrelated region

LINE

3`

5`

5`

3`

3`

5`

3`

5`

Transcription

Transcription

LINE-derived region

5`

5`

3`

3`

Transcript

Transcript

Reverse transcriptase

Translation

Recognition of the 3’-end sequence

5’

3`

5`

3`

Synthesis of cDNA

Synthesis of cDNA

3`

3`

5`

5`

3`

5`

3`

5`

New integration site

New integration site

slide26

Function of LINE Elements

1. Various Diseases

2. Evolutionary Change

3. Diversity & Phylogeny