Group I introns

Group II introns

- (some) encode RT, RNA maturase, endonuclease activities

- (some) encode “homing endonuclease”

Self-splicing introns (ribozymes) & mobile genetic elements

- retrohoming & retrotransposition

- for site-specific transposition

- in organellar genes of fungi, plants, protists;nuclear rRNA genes of certain protists & fungi, and (rarely) in bacteria & phage

- found in organelles of fungi, plants, protists;in bacteria & archaea (but rarely)

- two-step transesterification and lariat intron

& RNA maturase

- for splicing in vivo

Bonen & Vogel Trends Genet 17:322, 2001

Vicens & Cech Trends Biochem Sci 31: 2006

Chu RNA 4:1186, 1998

In vitro ribozymic splicing assay (in the absence of any protein)

Mutational analysis of branchpoint within domain VI of group II intron

- if branchpoint A, hydrolytic pathway (water acts as first acting nucleophile)

... and linear (rather than lariat) excised form of intron

Yeast mitochondrial aI5

Group II intronic ORFs encoding mobility & splicing functions

Bonen & Vogel Trends Genet 17:322, 2001

Topic 1 slide

(when discussing spliceosome catalysis being RNA vs. protein)

“Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase”

Dlakic & Mushegian RNA 17:799, 2011

“This is only the second example – the other one being telomerase - of the RT recruitment from a genomic parasite to serve an essential cellular function”

Diversity of “homing endonucleases” found in nature

Nuc, nuclear; cp, chloroplast; mt, mitochondrial; FS, free-standing genes; ARC, archaeal introns; GI, group I introns; GII, group II introns; INT, inteins

Hafez & Hausner Genome 55:553, 2012

HJR-like (resolvase homology); Vsr (patch-repair endonuclease homology)

Retroviruses

LTRRetrotransposons

Caulimoviruses

env

virus

LTRRetrotransposons

LTRs

gag int

virus

Hepadnavirus Non-LTR Retrotransposons

Group I I introns

Mitochondrialplasmid

RTL

msDNAs

intronmt

plasmidmt

genome

Bacterialgenome

RT-RH

Xiong & Eickbush, EMBO J 9:3353,1990

Evolutionary relationships among reverse transcriptase (RT) encoding elements

Group II introns are retroelements

Alberts Table 5-3

TRANSPOSABLE ELEMENTS

SINEs – short interspersed nuclear elements (eg Alu)

Cordaux & Batzer Nat Rev Genet 10:691, 2009

Transposable element (TE) content of human genome

Alu repeats: ~300 bp long with AluI restriction site, > 1 million copies in human genomeSVA composite

retroelement (SINE, VNTR & Alu)

VNTR: variable number tandem repeat

LINEs – long interspersed nuclear elements (eg L1)

By their sheer number and mobility, retrotransposons, DNA transposons and endogenous retroviruses have shaped our genotype and phenotype both on an evolutionary scale and on an individual level. Notably, at least the non-long terminal repeat retrotransposons are still able to cause disease by insertional mutagenesis, recombination, providing enzymatic activities for other mobile DNA, and perhaps by transcriptional overactivation and epigenetic effects. Currently, there are nearly 100 examples of known retroelement insertions that cause disease.

Solyom & Kazazian Genome Med 4:12, 2012

Identification of retroelement (L1) insertion events by monitoring variation among individuals

- human DNA sequence comparisons to determine number of non-reference mobile DNA insertions in Craig Venter’s genome

- extrapolated back to most recent common ancestor (using molecular clock) & estimated 1 in 21 people would have a new Alu, 1 in 212 would have a new L1, and 1 in 916 would have a new SVA

Xing et al. Genome Res 19:1516, 2009

PCR validation of MASV (mobile element-associated structural variants)

Craig Venter’s diploid genomePlos Biol 2007

How is transposon activity controlled by the host?

- DNA methylation status of promoter regions

- RNA interference (miRNAs/Argonaute proteins, piRNAs/Piwi proteins)

Transposons must be recognized as “foreign” & selectively silenced

Malone & Hannon Cell 136:656, 2009

Argonaute proteins are related to retrotransposase enzymes (Song et al. Science 305:1434, 2004)

Crystal structure Argonaute similar to RNase H domain (pol) of HIV

“The deeply conserved use of small RNAs as mechanisms to defend genomes against mobile elements points to this being a very early, or perhaps even the ancestral, role for RNAi-related pathways.”

Methods for detecting transposons insertions

O’Donnell & Burns Mobile DNA 1:21, 2010

Freschotte Nat Rev Genet. 3: 329, 2002

Plant LTR-retrotransposon movement can be triggered by stress

Detection of new genomic insertionsby transposon display technique

adaptor

ligate adaptors (linkers) to ends of restriction fragments

- look for extra PCR products

Transcription & mobilization in vivo

Wendel PNAS 97:6250, 2000

barley

southnorth

slopes only ~ 200 m

apart

Nevo PNAS 109: 2960, 2012

Active LTR-retrotransposon (BARE-1) in barley DNA

- genomic response to different microclimates

- examined BARE-1 copy number among barley populations found at top vs. bottom of “Evolution Canyon” in Israel (& on north vs. south sides of canyon)

Southern hybridization and PCR-based techniques to estimate the copy number of BARE-1 and the variability among individuals

south side drier than north side

BARE-1 comprises an average of 3% of the barley genome (range of 8,300 - 22,100 copies)

Kalendar PNAS 97: 6603 (2000) REMAP amplification with primers LTR-A and (CAC)7T

Observed marked variation in populations from adjacent locations

- recent transposition events

SSR = simple sequence repeat

North-Low (& South-Low): - less stress- lower BARE-1 copy number

Kalendar PNAS 97: 6603 (2000)

BARE-1 copy number at different locations in canyon

“The copy number of BARE-1 was correlated with aridity, suggesting a link between genome defense and stress responses. “

“... first significant step toward addressing McClintock’s challenge to figure out how cells restructure their genomes in response to perceived danger.”

in = integraseencoded withinBARE-1