Selfsplicing introns

1
Self-splicing introns

• Chris Templeton

2
Introduction

• Types of self-splicing introns
• Mechanics of self-splicing
• Mobility of self-splicing introns
• Relevant uses of these introns

3
Introns

• Genes have their coding sequences interrupted by
  non coding regions called introns.
• These introns must be cleaved to produce a
  functional protein, rRNA or tRNA
• Self-splicing introns can cleave themselves out
  of RNA

4
Self-splicing introns

• Discovered by Tom Cech in 1982 (Nobel Prize 1989)
• It showed that RNA could fold into complex shapes
• Catalyze biochemical reactions
• Provided the mechanistic details for RNA splicing

5
Types of self-splicing introns

• Group I
• Protozoa
• Fungal mitochondria
• Chloroplasts
• Bacteriophage T4
• Some bacteria
• Group II
• Chloroplasts
• Fungal mitochondria
• mRNA of some bacteria

6
Self-splicing introns

• Ribozyme
• An RNA molecule that catalyzes a chemical
  reaction
• They catalyze their own splicing
• They are self-splicing in vitro but usually
  require proteins in vivo.

7
Group I Splicing
5 splice site
3 splice site
Internal Guide sequence

• Occurs in two transesterification reactions
• Free guanosine attacks 5 splice site
• 5 exons 3-OH then attacks the 3 splice site

http//www.mun.ca/biochem/courses/3107/images/VVP/
Ch25/25-23.jpg
8
Group I intron

• G-Binding Site
• Conserved GC pair in P7

9
Converting Group I introns into Ribozymes

• A relinearized intron retains its active site
• 3 G-OH usually attacks a phosphate near the 5
  end (upstream of the IGS) in order to cyclize,
  but it has been removed so the intron remains
  linear.
• If G is provided the ribozyme will repeatedly
  catalyze cleavage
• The IGS can be changed in order to cleave diff.
  RNA

10
Group II Splicing

• Occurs in two transesterification reactions
• The 2-OH of a bulged A attacks the 5 splice
  site.
• The 3-OH of the 5exon attacks the 3 splice
  site.

http//www.mun.ca/biochem/courses/3107/images/Lodi
sh/Lod12-20.jpg
11
Group II intron
http//megasun.bch.umontreal.ca/cours/BCM-2002/Pub
lications/Introns-groupII-Bonen2001.pdf
12
Mobility of Group I Group II introns

• Catalytic intron RNA
• ORF (open reading frame) within the intron
• IEP (intron encoded protein)
• Stabilizes the catalytic active RNA structure
• RT activity
• Endonucleases
• Maturases (endonuclease/RT activity)

13
Maturases in yeast mtDNA

• Encoded in the ORF of the intron
• Splice the intron that encodes them
• Group I II maturases have a site-specific
  endonuclease activity that mediate intron
  mobility.

14
Mobility of Group II introns

• After translation the maturase associates with
  the excised intron to form a Ribonucleoprotein
  (RNP) complex
• Group II introns can be inserted directly into
  target sites in double stranded DNA
• IEP that has RT, RNA splicing and endonuclease
  activity mediates the process

http//megasun.bch.umontreal.ca/cours/BCM-2002/Pub
lications/Introns-groupII-Bonen2001.pdf
15
Mobility of Group II introns

• Introns achieve site specific insertion (homing)
  via endonucleases encoded within the intron
• Each intron-encoded endonuclease cleaves at a
  different target sequence
• The intron is inserted into intron- alleles.

http//megasun.bch.umontreal.ca/cours/BCM-2002/Pub
lications/Introns-groupII-Bonen2001.pdf
http//www.sciencemag.org/cgi/reprint/289/5478/452
.pdf
16
Reverse splicing
http//www.sciencemag.org/cgi/reprint/289/5478/452
.pdf

• Has been demonstrated in vitro for Group I and
  Group II introns
• Ribonucleoprotein (RNP) complex recognizes
  specific sequences
• Intron binds to the DNA using its 3-OH tail

http//megasun.bch.umontreal.ca/cours/BCM-2002/Pub
lications/Introns-groupII-Bonen2001.pdf
17
Reverse splicing

• The opposite strand is cleaved
• The 3 end of the cleaved strand is used as a
  primer to reverse transcribe the inserted intron
  RNA
• The spliced RNA intron serves as a template for
  RT
• The single stranded cDNA is then incorporated by
  recombination or repair mechanisms

http//megasun.bch.umontreal.ca/cours/BCM-2002/Pub
lications/Introns-groupII-Bonen2001.pdf
18
Retargeting introns to specific sites

• Group II introns can be retargeted to different
  regions in order for gene therapy
• For example
• CCR5 the co-receptor on T-cells which is used by
  HIV-1
• Individuals with mutations in CCR5 are resistant
  to HIV-1 and have no other pathologies

19
Group II introns designed to insert into
therapeutically relevant DNA target sites in
human cells

• Huatao Guo, et al.
• Science 289, 452 (2000)

20
Human therapy uses

• HIV-1 provirus DNA and CCR5 have been targeted in
  order to disrupt their synthesis
• The exon binding sites on the intron were
  modified and a new sequence for the IEP was
  developed

21
Retargeting of intron

• All of the retargeted introns inserted at
  precisely the correct position in HIV-1 and CCR5
  (confirmed by sequencing)
• Intron insertion frequency ranged from 0.16 to
  66

22

• PCR, restriction enzyme analysis and sequencing
  confirmed that the retargeted introns had
  inserted at the correct locations

http//www.sciencemag.org/cgi/reprint/289/5478/452
.pdf
23
Results

• Group II introns can be retargeted to insert
  efficiently into desired DNA target sites
• The RNP does retain activity in human cells
• Enrich of the most efficient introns to use for
  experiments
• This technique could possibly be used for other
  diseases

24
Conclusion

• Group I II introns are ribozymes
• RNA intron forms a 3 structure which allows them
  to self splice
• The spliced intron can retrohome into specific
  sites using sequences on intron and Maturase
• This technique can in the future be used for
  human therapy, genetic engineering and genetic
  knockout

25
References

• Bonen, L., Vogel, J, Trends in Genetics Vol. 17
  No. 6 2001
• Cech, T. Annu. Rev. Biochem. 1990. 59543- 68
• Saldanha, R., et al. FASEB Journal Vol. 7 1993
• Lambowitz, A., Zimmerly, S. Annu. Rev. Genet.
  2004. 381-35
• Molecular Biology of he Gene 5th ed.
• http//www.mun.ca/biochem/courses/3107/Topics/Spli
  cing.html

26
Questions