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
Mitochondria and plastid genomes of plants and protists (rRNA, tRNA and mRNA genes).
Nucleus of certain protists, fungi and lichens, but only rRNA genes.
Eubacteria (tRNA genes) & phages.
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 Intron-minus Intron-plus Cleavage Enase ORF
Recombination
Intron-plus
Enase ORF
Homing Enase
Enase - endonuclease

10. A type of homologous recombination.
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 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. tRNA splicing in two enzymatic steps
tRNA splicing in two enzymatic steps. Group II and NmRNA splicing related.

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 (Odom et al. 2004) 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
Group II Intron (with its protein) from an Antarctic Chlamydomonas raudensis. The intron-ORF phylo. tree shows unrelated organisms grouping together [Chlamydomonas, Euglena (E. myxocylindracea), and Nostoc (cyanobacteria)], indicative of horizontal transfers of this intron.
(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.
Mostly from studies of a bacterial (Lactococccus lactis) group II intron by the Belfort and Lambowitz labs.

24. Intron Loss by Reverse Transcription and Recombination
from Lambowitz and Belfort, 1993

25. Reverse transcriptase activity of the psbA1 intron-encoded protein
Assay with polyrA/oligodT; RT spec. activity= pmoles/min/pmole protein,
Similar to AMV ( w/same units)

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

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?