Group I Introns

1
Group I Introns

• Distribution Structure
• Splicing Mechanism Regulation
• Intron Homing
• Intron-Encoded Endonucleases

2
Distribution of Group I introns

• Prokaryotes eubacteria (tRNA rRNA), phage
• Eukaryotes
• lower (algae, protists, fungi)
• nuclear rRNA genes, organellar genes, Chlorella
  viruses
• higher plants organellar genes
• lower animals (Anthozoans) mitochondrial
• gt1800 known, classified into 12 subgroups,
  based on secondary structure

3
Cr.psbA4 Secondary Structure
3D Model of Tt.LSU Intron
Michel and Westhof, JMB, 1990
4
Group I Introns are Ribozymes
Self-Splicing Mechanism
GOH, guanosine In vivo, GTP.
3 terminal G is nearly 100 conserved.
5
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 ()
6
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 and vertical.
7
psbA encodes D1 polypeptide of PSII
Yamamoto, Plant Cell Physiol., 2001
D1 protein turns over rapidly because it becomes
damaged in bright light.
8
Light-Dependent Splicing of psbA
psbAi2 intron
LSU rRNA intron
9
Light-Stimulated Splicing of psbA

• Is coordinate splicing of all 4 introns
  increases gt 6-8-fold
• Involves photosynthetic electron transport
• Necessary to maintain autotrophic growth rates

10
Efficient splicing of chloroplast group I
introns in vivo probably requires trans-acting
proteins.
Strategy to identify them Suppressor
Genetics 1. Replace the Cr. LSU rRNA intron
with a version that self-splices poorly in
vitro, and limits growth in vivo.2. Isolate
revertents whose Cr.LSU splicing is
substantially restored.3. Perform genetic
analysis to identify extragenic suppressors.4.
Clone extragenic suppressor genes.
11
Cr. LSU Intron Replacement Plasmid
12
RNA Analysis of Suppressors
13
The rRNA intron suppressors also suppress a
similar deficiency in a psbA intron (i4).
14
Genetic Analysis of the Suppressors

• 3 distinct nuclear genes css1, css2 and css3
  - (css1 and css3 are linked)
• css1 and css2 are dominant
• All 3 genes promote splicing of the rRNA and
  psbA4 introns.

15
Group I intron homing
16
Homing Endonucleases (HEs)

• Site- specific endonucleases
• Recognize long DNA targets 20-40 bp
• 4 families based on a conserved catalytic motif
• 1. LAGLI-DADG
• 2. GIY-YIG
• 3. H-N-H
• 4. His-Cys
• Mobilizing elements
• mobile Group I introns
• as inteins (inframe protein insertions)
• mobile HEs

17
Mobile Introns in C. reinhardtii

• LSU rRNA - ORF encodes I-CreI
• psbA2 - ORF encodes ??
• psbA4 - ORF encodes I-CreII

18
Structure of psbA Intron 4 ORF
19
I-CreII Over-Expression Purification
20
In vitro Cleavage Assay
I-CreII
2.1 kb
pE4E5 4 kb
1.9 kb
Sca I
21
Determining the boundaries of the Recognition
Sequence
22
I-CreII Recognition Cleavage Sites
5 AACATTGTAGCTGC TCATGGTTACTTTGG 3 3
TTGTAACATCGA CGAGTACCAATGAAACC 5
Intron 4
Kim et al. (2005)
23
Cleavage of Heterologous psbA DNAs
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25
Yijia Bao Laura Corina Nita Deshpande Stephen
Holloway Hyong-Ha Kim Jaesung Lee Fei Li O.W.
Odom Andrew Thompson Jinshan Wang Xiaoqui Yuan

Current lab members
This research has been supported primarily by
USDA, DOE, and Robert Welch Foundation.