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

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

Anabaena T4 phageMetridium

Distribution of Group II introns is a little more restrictive

1. Mitochondrial and plastid genomes of plants and protists (rRNA, tRNA and mRNA genes)

2. Eubacteria (mRNA, most between genes)

3. Archae

4. Metazoan mitochondria

• Not found in nuclear or viral genes

Methanosarcina

Nephtys

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.

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)

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.

Is there anything about these introns (group I or II) that would support their suggested tendency for horizontal transfer and integration into genes?

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

Group I intron homingGroup I intron homing

Intron-plusIntron-plus

Enase ORFEnase ORF

Homing EnaseHoming Enase

Intron-Intron-minusminus

CleavageCleavage

Intron-plusIntron-plus

RecombinationRecombination

Enase - endonuclease

DSBR Modelfor Group I Intron Homing

From Lambowitz and Belfort(1993).

A type of homologous recombination.

In+

In-

4 families of 4 families of homing endonucleaseshoming endonucleases (based on the (based on thepresence of a conserved catalytic motif):presence of a conserved catalytic motif):

1. LAGLIDADG1. LAGLIDADG

2. GIY-YIG2. GIY-YIG

3. H-N-H3. H-N-H

4. His-Cys4. 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

I-CreI bound to DNA

Structure and Splicing of Group I and Group II introns:

1. Have different, but conserved structures– many subfamilies of group I and II introns

2. Splice by different mechanisms

3. Many are capable of self-splicing (i.e., no proteins required, the RNA itself is catalytic, a "ribozyme")

4. Proteins facilitate splicing in vivo

Cr.LSU intron: 2ndary structure of a group I intron

Old style drawing Newer representation

Conserved core

5’ splice site

Exon seq. in lower case and boxedShows how splice sites can be brought close together by “internal guide sequence”.

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.

Splicing mechanism for group I introns

IVS – intronGOH - GTP

Last nt of intron is always a G !!

Guanosine binding site of Group I Introns

1. It is mainly the G of a G-C pair in the P7 helix of the conserved core

- forms a triple base pair

2. It is highly specific for Guanosine (Km ~20 μM).

3. Binds free GTP in the first splicing step.

4. Binds the 3’-terminal G of the intron in the second splicing step.

Protein (splicing) factors for group I introns

• 2 types:1. Intron-encoded (promote splicing of only the

intron that encodes it), called Maturases2. Nuclear-encoded (for organellar introns)

• Nuclear-encoded ones function by:1. Promoting correct folding of the intron (e.g., CBP2

promotes folding of a cytochrome b intron)2. Stabilizing the correctly folded structure (cyt18

promotes activity of a number of group I introns) • Cyt18 is also the mitochondrial tyrosyl-tRNA

synthetase

Consensusstructure of group II introns

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 BarkanU. Oregon

Lake Bonney

McMurdo Dry valley, Antartica

Glacier

John Priscu et al.

Domains of the psbA1 ORF:RT - reverse transcriptase

(subdomains 0-7)X - maturaseD - DNA-binding

HNH - endonuclease

Phylogenetic analysis places it in group IIB2 intron ORFs

A group II intron ORF

(Odom et al. 2004)

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

from Lambowitz and Belfort, 1993

Intron Loss by Reverse Transcription and Recombination

0

1 104

2 104

3 104

4 104

5 104

6 104

7 104

0 10 20 30 40 50

y = 3084.4 + 2892.1x R= 0.99921 y = 3021.9 + 902.37x R= 0.99903

25oC

37oC

Time, min

[32P

]dTT

P in

corp

orat

ed, c

pm

Time course of RT activity of fusion protein at 37oC and 25oC

in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,

5 mM DTT, 75mM NaCl, 25 mM KCl

A

B

Reverse transcriptase activity of the psbA1 intron-encoded protein

Is there anything about these introns (group I or II) that would support their suggested tendency for horizontal transfer and integration into genes?

1.Both groups contain homing introns.2.A bacterial (Lactococcus) Group II intron (Ltr) has been shown to jump to new sites.3.If an intron can promote its own splicing, then its less likely to disrupt a gene when it inserts. 4.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.

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

Box 6.7 (Buchanan et al.)

tscA RNA also required, part of 1st intron

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.

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?