Intron Classes

1
Intron Classes Distribution

• Group I - organelles, nuclear rRNA genes of
  certain eukaryotes, bacteria (gt1500)
• Group II - organelles, bacteria, archaea (gt
  1000)
• Nuclear mRNA (NmRNA) - ubiquitous in eucaryotes
• Nuclear tRNA- some eucaryotes

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Self-Splicing Introns

• Some Group I and Group II introns can
  self- splice in vitro in the absence of proteins.
• Each group has a distinctive, semi-conserved
  secondary structure.
• Both groups require Mg2 to fold into a
  catalytically active ribozyme.
• Group I introns also require a guanosine
  nucleotide in the first step.

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Protein factors forGroup I Group II Introns

• 2 types (based on genetic location)
• Intron-encoded
• - promotes splicing of the intron that encodes
  it
• Nuclear-encoded
• - for organellar introns
• - identified in fungi (for mito. introns)

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Evolutionary Intron Dynamics

• Intron gain
• Intron loss
• Intron transposition
• Horizontal transfer
• Evidence for horizontal transfer is not uncommon
  for these introns
• Unrelated introns in the same genetic location in
  clearly related organisms.
• Similar introns in completely unrelated genes
  organisms.

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Intron Homing

• the invasion of an intron-minus gene/allele by
  the intron from an intron-plus allele.
• Catalyzed by a protein encoded by the mobile
  intron.

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Group I intron homing
Enase - endonuclease
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Group I intron-containing genes in Chlamydomonas
reinhardtii cpDNA
psbA
psbA


(4)
5s
5s
203 kb
IR
IR
23s
23s


(1)
16s
16s
Indicates group I intron and ()
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psbA gene of Chlamydomonas reinhardtii
1. All 4 introns self-splice in vitro. 2. ORFs do
not seem to encode maturases. 3. Introns 2 and 4
are mobile. 4. Likely have multiple origins
horizontal ( i3) and vertical (i4). Intron
4 is found in anciently diverged Chlamydomonas
spp. Intron 3 is most similar to an intron in
bacteriophage T4.
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Mobile Introns in C. reinhardtii

• LSU rRNA - ORF encodes I-CreI
• psbA2 - ORF encodes ??
• psbA4 - ORF encodes I-CreII
• psbA4 is very efficient at homing because
• I-CreII has its own promoter
• I-CreII is a fairly robust enzyme
• There is a recombination hot spot at the end exon
  5.

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DNA cleavage by I-CreII
I-CreII
2.1 kb
pE4E5 4 kb
1.9 kb
Sca I
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Determining the boundaries of the Recognition
Sequence
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I-CreII Cleavage Recognition Sites
5 ACAACATTGTAGCTGC TCATGGTTACTTTGGTC 3 3
TGTTGTAACATCGA CGAGTACCAATGAAACCAG 5
Intron 4
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Cleavage of Heterologous psbA DNAs
Advantage promote homing even if the target
changes by a few nucleotides.
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Red Snow
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Chlamydomonas nivalis (augustae)

• Most common snow alga of Northern Hemisphere
  Red Snow is the aplanospore form
• Red pigment is Astaxanthin (photoprotective
  carotenoid)
• Resistant to UV light
• Usually closely associated with bacteria

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• Do ribozymes show evidence of biochemical
  adaptation to low temperature?
• Ribozymes have not been characterized from
  psychrophiles.

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Many organisms are highly adapted to
environmental temperature

• Psychrophiles (lt 20?C)
• Mesophiles ( 20-35?C)
• Thermophiles ( 35-70?)
• Hyperthermophiles ( gt 70-110?C)

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Protein enzymes show evidence of adaptation to
low temperature

• Enzymes from psychrophilic organisms
• usually have a lower temperature optimum than
  their mesophilic counterpart
• are typically more thermolabile
• are usually more efficient catalysts
• are likely more flexible

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Cn.psbA preRNA catalyzes a reaction with GTP
that resembles the first splicing step. 2nd
step, exon ligation, was not detected.
301 nts
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Cn.LSU self-splices in vitro.
Conditions 200 ?M GTP 25 mM MgCl2 pH 7.5
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Adaptation in the psychrophiles intron ribozymes?

• The C. nivalis homologues are much smaller than
  the reinhardtii introns (lack the ORFs), and are
  a little more A-T rich.
• Self-splicing of the C. nivalis rRNA intron
  (Cn.LSU) is more thermosensitive than that of
  Cr.LSU.
• The temperature optima for self-splicing of the
  Cn.LSU and Cr.LSU introns are similar.
• The Cn.psbA intron does not self-splice, though
  it is autocatalytic.

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McMurdo Dry valley, Antartica
Glacier
Lake Bonney
John Priscu et al.
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Chlamydomonas CCMP-1619

• Psychrophilic grows only lt 20?C
• Adapted to constant dim light and low
  turbulence.
• It is not Chlamydomonas subcaudata, as
  originally thought.
• A variety of Chlamydomonas raudensis?
• Lacks typical group I introns (homologues of
  Cr.psbA4 ( Cr.psbA3) and Cr.LSU)
• Contains a Group II intron in exon 4 equivalent.

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Cra.psbA1
- most similar to introns from Calothrix and
Nostoc - unique insertion in domain I - ORF in
domain IV
2.5 kb
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Domains of the psbA1 ORF RT - reverse
transcriptase X - maturase D - DNA-binding HNH -
endonuclease
Phylogenetic analysis of the ORF places it in
chloroplast-like Class 2 (S. Zimmerly) or
subgroup IIB2 (F. Michel).
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Self-splicing of Cra.psbA1 pre-RNAs
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Temperature-dependence of self-splicing of psbA1
pre-RNAs
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Splicing of the psbA1 intron in E. coli is
ORF-dependent
FS - frame-shift mutation at amino acid 10 PM
point mutations (1 in RT, 1 in maturase domain)
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A
B
34
from Lambowitz and Belfort, 1993
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A Possible Evolutionary Path for an Ancient
Mobile psbA Intron

Mobile Self-splicing Intron (C. reinhardtii
C. moewussi) Intron with degenerate ORF (C.
subcaudatum) Non self-splicing intron (C.
nivalis) that lacks an ORF Intron
lost (C. raudensis)
Group II intron
36
O.W.
J.G.
S.C.
D.S.
D.L.H.
J.P.
O.W. Odom, Dave Shenkenberg, Josh Garcia, Jangwon
Park, Samantha Croft