Organellar Introns

1
Organellar Introns

• Organellar genomes contain 3 types of introns
• 1. Group I
• 2. Group II (evolutionary precursors to nuclear
  mRNA/spliceosomal introns)
• 3. Group III (related to Group II introns,
  common in Euglenoids)
• - Twintrons, intron inserted into an intron

2
Distribution of Group I introns is broad but
weirdly irregular

1. Mitochondria and plastid genomes of plants and
   protists (rRNA, tRNA and mRNA genes).
2. Nucleus of certain protists, fungi and lichens,
   but only rRNA genes.
3. Eubacteria (tRNA genes) phages.
4. Metazoans - only in mitochondrial genes of a few
   anthozoans (e.g., sea anemone).

Tetrahymena
T4 phage
Anabaena
Metridium
3
Distribution of Group II introns is a little more
restrictive

• Mitochondrial and plastid genomes of plants and
  protists (rRNA, tRNA and mRNA genes)
• Eubacteria (mRNA, most between genes)
• Archae
• Metazoan mitochondria
• Not found in nuclear or viral genes

Methanosarcina
Nephtys
4

• Evidence for horizontal transfer is common for
  these introns
• Same gene in related organisms with different
  introns (in the same positions).
• Same, or similar introns found in completely
  unrelated genes organisms.
• Phylogenetic (or reconstruction) analysis also
  supports their having been constantly lost and
  gained during evolution.

5
psbA gene of Chlamydomonas reinhardtii has 4
group I introns of vertical and horizontal origins
Intron 4 is found in anciently diverged
Chlamydomonas spp. - acquired vertically Intron
3 is most similar to an intron in bacteriophage
T4 - may have been acquired horizontally
(Holloway et al. 1999)
6
A degenerate form of Intron 3 (psbA) lies between
the petA and petD genes of cpDNA
Possibly intron 3 inserted here between 2 genes,
and then degenerated over time because splicing
was not necessary.
7
Is there anything about these introns (group I or
II) that would support their suggested tendency
for horizontal transfer and integration into
genes?
8
Intron Homing

• Has been demonstrated experimentally for both
  group I group II introns
• It is the invasion of an intron-minus allele by
  the intron from an intron-plus allele.
• result is conversion of the intron-minus allele
  to intron-plus.
• Initiated by a protein encoded by the mobile
  intron

9
Group I intron homing
Enase - endonuclease
10
DSBR Model for Group I Intron Homing
In-
In
A type of homologous recombination.
From Lambowitz and Belfort (1993).
11
4 families of homing endonucleases (based on
the presence of a conserved catalytic motif)
I-CreI bound to DNA
1. LAGLIDADG 2. GIY-YIG 3. H-N-H 4. His-Cys

• Recognize long DNA sequences 20-40 bp (cut
  rarely in large genomes)
• Tolerate mutations in the recognition sequence
• Exist outside of introns (and are also mobile
  elements)
• Have invaded GI introns, thereby mobilizing them
  

12
Structure and Splicing of Group I and Group II
introns

• Have different, but conserved structures
• many subfamilies of group I and II introns
• Splice by different mechanisms
• Many are capable of self-splicing (i.e., no
  proteins required, the RNA itself is catalytic,
  a "ribozyme")
• Proteins facilitate splicing in vivo

13
Cr.LSU intron 2ndary structure of a group I
intron
Old style drawing
Newer representation
Exon seq. in lower case and boxed
Shows how splice sites can be brought close
together by internal guide sequence.
Conserved core
5 splice site
14
3-D Model of Tetrahymena rRNA Intron
Catalytic core consists of two stacked helices
domains 1. P5 P4 P6 P6a (in green) 2. P9
P7 P3 P8 (in purple) The substrate is
the P1 P10 domain (in red and black), it
contains both the 5 and 3 splice sites.
15
Splicing mechanism for group I introns IVS
intron GOH - GTP Last nt of intron is always a
G !!
16
Guanosine binding site of Group I Introns

• It is mainly the G of a G-C pair in the P7 helix
  of the conserved core
• - forms a triple base pair
• It is highly specific for Guanosine (Km 20 µM).
• Binds free GTP in the first splicing step.
• Binds the 3-terminal G of the intron in the
  second splicing step.

17
Protein (splicing) factors for group I introns

• 2 types
• Intron-encoded (promote splicing of only the
  intron that encodes it), called Maturases
• Nuclear-encoded (for organellar introns)
• Nuclear-encoded ones function by
• Promoting correct folding of the intron (e.g.,
  CBP2 promotes folding of a cytochrome b intron)
• Stabilizing the correctly folded structure (cyt18
  promotes activity of a number of group I introns)
  
• Cyt18 is also the mitochondrial tyrosyl-tRNA
  synthetase

18
(No Transcript)
19
Consensus structure of group II introns
20
Angiosperm chloroplast introns

• 16 group II introns
• 1 group I intron (leutRNA), descended from a
  cyanobacterial leutRNA (tRNA L) intron
• Splicing factors for the group II introns
• Most are nuclear-encoded (A. Barkan)
• At least one is intron-specific
• Others splice a group of introns
• Some are PPR (pentatricopeptide repeat) proteins
• Helical proteins that bind macromolecules (RNA
  and proteins)
• 1 factor is intron-encoded in the lystRNA (tRNA
  K) intron, a.k.a. maturase K (or matK)

Alice Barkan U. Oregon
21
McMurdo Dry valley, Antartica
Glacier
Lake Bonney
John Priscu et al.
22
A group II intron ORF
Domains of the psbA1 ORF RT - reverse
transcriptase (subdomains 0-7) X - maturase D -
DNA-binding HNH - endonuclease
Phylogenetic analysis places it in group IIB2
intron ORFs
(Odom et al. 2004)
23
Group II Intron Homing (retrohoming pathway)
Spliced intron RNA (with bound protein, RT)
reverse splices into sense strand of DNA target.
Protein cuts anti-sense strand in the 3 exon
(exon 2). Protein reverse transcribes RNA,
making cDNA copy of intron RNA. Repair synthesis
replaces RNA with DNA, ligates DNAs.
www.fp.ucalgary.ca/group2introns/mobility.htm
24
Intron Loss by Reverse Transcription and
Recombination
from Lambowitz and Belfort, 1993
25
Reverse transcriptase activity of the psbA1
intron-encoded protein
A
B
26

• Is there anything about these introns (group I or
  II) that would support their suggested tendency
  for horizontal transfer and integration into
  genes?
• Both groups contain homing introns.
• A bacterial (Lactococcus) Group II intron (Ltr)
  has been shown to jump to new sites.
• If an intron can promote its own splicing, then
  its less likely to disrupt a gene when it
  inserts.
• Could potentially move multiple ways at the DNA
  level, or at the RNA level by reverse splicing
  into another RNA, which gets copied into DNA by
  the RT and recombines into the genome.

27
TRANS-splicing

• A few cp and mitochondrial mRNAs are formed by
  trans-splicing
• - separate RNAs are joined together
• - still contain intron-exon organization
• - introns contain Group II consensus sequences
• Examples
• - rps12 in tobacco 5' and 3'-halves are
  encoded at separate sites on cpDNA
• - psaA in Chlamydomonas three exons, each is
  encoded at separate sites, maturation requires
  2 trans-splicing events

28
(No Transcript)
29
tscA RNA also required, part of 1st intron
Box 6.7 (Buchanan et al.)
30
Splicing of the first psaA intron involves 3
RNAs! One, tscA, is internal to in the intron,
and contains part of Domain 1, all of Domains 2
and 3, and part of Domain 4. tscA is encoded as
a separate gene co-transcribed with chlN gene.
31
Trans-acting Factors for Trans-Splicing

• Trans-splicing of the psaA1 introns in
  Chlamydomonas requires a large number of
  nuclear genes (at least 14)
• 3 of these genes have been cloned proteins
  reside (at least in part) in a large RNP
  (ribonucleoprotein particle)
• Evolutionary intermediate between group II
  introns and nuclear mRNA introns?