Transposition and L1 Retrotransposons

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Transposition and L1 Retrotransposons

• Raleigh Parrott

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Transposition

• Genetic recombination process
• Utilizes transposons
• Excision/ Insertion (Cut and Paste Mechanism)
• Duplication/ Insertion (Replicative Mechanism)
• RNA intermediate
• Described as a random insertion

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
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Transposition
New Site
Old Site
Excision/ Insertion
Duplication/ Insertion
Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
4
Transposable Elements

• Transposons in all genomes
• Humans
• 2 of DNA sequence encodes for cellular proteins
• gt50 is transposon related
• Transposon content varies between genomes
• Transposition regulation

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
5
Transposon Classes

• DNA Transposons
• LTR (long terminal repeats) Retrotransposons
  (Viral)
• Poly-A Retrotransposons (Non-viral)

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
6
DNA Transposons
Terminal Inverted Repeats
Element
Transposase
Host DNA
Target Site Duplication

• Recombination sites are the Terminal Inverted
  Repeats
• Each transposon uses its own transposase for
  recombination
• Target site duplications are generated during
  recombination

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
7
LTR Retrotransposons (Viral)
Element
Integrase and Reverse Transcriptase
Target Site Duplication
LTR (Long Terminal Repeat)

• Recombination sites are the LTR sequences
• Reverse Transcriptase DNA polymerase (RNA to
  DNA)
• Integrase enzyme to integrate DNA into novel
  site

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
8
Poly-A Retrotransposons
3 UTR
5 UTR
ORF 1
ORF 2
Poly-A Sequence

• Do NOT have terminal inverted repeats
• Have untranslated regions (UTR), the 3 UTR being
  followed by poly-A
• ORF-1 is a gene encoding an RNA-binding protein
• ORF-2 encodes a protein with both reverse
  transcriptase and endonuclease activity

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
9
Poly-A Mechanism
LINE DNA
Transcription via RNA polymerase
LINE mRNA

• Translation
• Bind ORF 1 and 2 proteins

AAAA
Target DNA
TTTT

• Binding to target DNA
• Target site cleavage
• RNA-DNA hybrid formation

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Poly-A Mechanism
cDNA synthesis

• RNA Degradation second strand synthesis
• DNA joining/ repair

AAAA
TTTT
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Poly-A Mechanism

• Called target site primed reverse transcription
• The ORF 1 and 2 proteins remain associated with
  the encoding mRNA
• cDNA copied DNA
• LINE Long Interspersed Nuclear Element

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
12
LINE Transposition

• Compose 20 of human genome
• L1 is the best understood
• Promote own mobility
• Also donate proteins needed for SINEs
  (nonautonomous)
• SINE Short Interspersed Nuclear Elements
• Appear as simple genes

Watson, J.D. Baker, T.A. Bell, S.P. Gann, A.
Levine, M. Losick, R. Site-Specific
Recombination and Transposition of DNA. In
Molecular Biology of the Gene, 5th Ed. Pearson
San Francisco, 2004 310-337.
13
L1 Retrotransposons and DNA Damage

• DNA damage can affect retrotransposition
• Regulation of L1 transcription
• L1 insertion into pre-existing DNA breaks
• Alterations in DNA repair machinery

Farkash, E.A, Luning Prak, E.T. Journal of
Biomedicine and Biotechnology, 2006, Article ID
37285, 1-8.
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L1 Retrotransposons and DNA Damage

• Transcriptional Regulation
• Decrease L1 mRNA levels
• Alter transcription factor levels or binding
  affinity (methylation)
• L1 Insertion
• Inactive L1 can enter upon a double strand
  break (endonuclease independent)
• DNA Repair Machinery
• DNA repair proteins can affect the mobility of
  retrotransposons

Farkash, E.A, Luning Prak, E.T. Journal of
Biomedicine and Biotechnology, 2006, Article ID
37285, 1-8.
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L1 Activation and Genotoxic Stress

• May be beneficial
• Stabilize double strand breaks (DSBs)
• Possible specialized DNA repair??
• May be harmful
• Random insertion
• Endonuclease production
• Without integration, translocation and
  chromosomal rearrangements
• Both cell cycle arrest and apoptosis accompany
  retrotransposition in stressed cells

Farkash, E.A, Luning Prak, E.T. Journal of
Biomedicine and Biotechnology, 2006, Article ID
37285, 1-8.
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L1 Retrotransposition and Cancer

• In normal somatic cells, retroelement activity is
  repressed
• In cancer cells, retroelement activity seems to
  be somewhat reactivated
• Hypomethylation, transcripts and protein products
  are detected, L1 sequences at recombination sites
• However, the safeguards against
  retrotransposition remain intact
• Cancer cells may be vulnerable as well

Schulz, W.A. Journal of Biomedicine and
Biotechnology, 2006, Article ID 83672, 1-12.
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Role of Histone H2AX

• A double strand break (DSB) causes
  phosphorylation of H2AX (?-H2AX)
• ?-H2AX flanks DSB to promote cell survival
• Provides platform for other damage proteins
• H2AX activated in L1 expressing cells

Belgnaoui, S.M. Gosden, R.G. Semmes, O.J.
Haoudi, A. Cancer Cell International, 2006, 6,
13-22.
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L1 and Cancer

• L1 has destabilizing genome effects
• Accumulation of ?-H2AX
• Induction of apoptosis 7x more than
  mock-transfection
• L1 activation combined with radiation may lead to
  more selective apoptosis
• Radiation of cancer cells causes DSBs
• L1 competes with repair processes
• Causes overall genome destabilization leading to
  apoptosis

Belgnaoui, S.M. Gosden, R.G. Semmes, O.J.
Haoudi, A. Cancer Cell International, 2006, 6,
13-22.
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RNAi

• Sequence specific posttranscriptional gene
  silencing method
• Uses small interfering RNA sequences to target
  complementary RNA sequences for destruction
• Need dsRNA sequences

Horman, S.R. Svoboda, P. Luning Prak, E.T.
Journal of Biomedicine and Biotechnology, 2006,
Article ID 32713, 1-8.
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Possible L1 dsRNA Sources
Horman, S.R. Svoboda, P. Luning Prak, E.T.
Journal of Biomedicine and Biotechnology, 2006,
Article ID 32713, 1-8.
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Silencing L1 via RNAi

• Possible routes
• Decreases amount of L1 RNA
• RNAi machinery methylates L1 DNA to silence
  insertions
• RNAi alters chromatin accessibility (limits L1
  activity)

Horman, S.R. Svoboda, P. Luning Prak, E.T.
Journal of Biomedicine and Biotechnology, 2006,
Article ID 32713, 1-8.
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Current RNAi Efforts

• Demonstration of siRNAs from L1 elements
• How is L1 dsRNA assembled?
• Perturbing RNAi components and review changes in
  L1 retrotransposition frequency

Horman, S.R. Svoboda, P. Luning Prak, E.T.
Journal of Biomedicine and Biotechnology, 2006,
Article ID 32713, 1-8.
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In Summary

• L1 Retrotransposons as DNA repair?
• Use of L1 retrotransposons in cancer treatment?
• How does the body regulate L1? RNAi?