Group I introns

1. Self-splicing introns (ribozymes) & mobile genetic elements Group I introns - in organellar genes of fungi, plants, protists; nuclear rRNA genes of certain protists & fungi, and (rarely) in bacteria & phage - (some) encode “homing endonuclease” - for site-specific transposition & RNA maturase - for splicing in vivo Vicens & Cech Trends Biochem Sci 31: 2006 Group II introns - found in organelles of fungi, plants, protists; in bacteria & archaea (but rarely) - (some) encode RT, RNA maturase, endonuclease activities - retrohoming & retrotransposition - two-step transesterification and lariat intron Bonen & Vogel Trends Genet 17:322, 2001

2. Mutational analysis of branchpoint within domain VI of group II intron Yeast mitochondrial aI5g In vitro ribozymic splicing assay (in the absence of any protein) - if D branchpoint A, hydrolytic pathway (water acts as first acting nucleophile) ... and linear (rather than lariat) excised form of intron Chu RNA 4:1186, 1998

3. Group II intronic ORFs encoding mobility & splicing functions “Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encodedreverse 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” Topic 1 slide (when discussing spliceosome catalysis being RNA vs. protein) Bonen & Vogel Trends Genet 17:322, 2001

4. 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 HJR-like (resolvase homology); Vsr (patch-repair endonuclease homology) Hafez & Hausner Genome 55:553, 2012

5. gag RT-RH int LTRs virus env Hepadnavirus Non-LTR Retrotransposons Bacterial genome virus intron mt plasmid mt genome LTR Retrotransposons Retroviruses Caulimoviruses msDNAs LTR Retrotransposons Mitochondrial plasmid Group I I introns RTL Evolutionary relationships among reverse transcriptase (RT) encoding elements Group II introns are retroelements Xiong & Eickbush, EMBO J 9:3353,1990

6. TRANSPOSABLE ELEMENTS Alberts Table 5-3

7. Transposable element (TE) content of human genome LINEs – long interspersed nuclear elements (eg L1) SINEs – short interspersed nuclear elements (eg Alu) Alu repeats: ~300 bp long with AluI restriction site, > 1 million copies in human genome SVA composite retroelement (SINE, VNTR & Alu) VNTR: variable number tandem repeat 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 Cordaux & Batzer Nat Rev Genet 10:691, 2009

8. Craig Venter’s diploid genome Plos Biol 2007 Identification of retroelement (L1) insertion events by monitoring variation among individuals PCR validation of MASV (mobile element-associated structural variants) - 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

9. How is transposon activity controlled by the host? Transposons must be recognized as “foreign” & selectively silenced - DNA methylation status of promoter regions - RNA interference (miRNAs/Argonaute proteins, piRNAs/Piwi proteins) Argonaute proteins are related to retrotransposase enzymes (Song et al. Science 305:1434, 2004) “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.” Crystal structure Argonaute similar to RNase H domain (pol) of HIV Malone & Hannon Cell 136:656, 2009

10. Methods for detecting transposons insertions O’Donnell & Burns Mobile DNA 1:21, 2010

11. Plant LTR-retrotransposon movement can be triggered by stress Detection of new genomic insertions by transposon display technique Transcription & mobilization in vivo ligate adaptors (linkers) to ends of restriction fragments adaptor - look for extra PCR products Freschotte Nat Rev Genet. 3: 329, 2002

12. barley Active LTR-retrotransposon (BARE-1) in barley DNA - 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) - genomic response to different microclimates south north slopes only ~ 200 m apart south side drier than north side Nevo PNAS 109: 2960, 2012 BARE-1 comprises an average of 3% of the barley genome (range of 8,300 - 22,100 copies) Southern hybridization and PCR-based techniques to estimate the copy number of BARE-1 and the variability among individuals Wendel PNAS 97:6250, 2000

13. SSR = simple sequence repeat Observed marked variation in populations from adjacent locations - recent transposition events REMAP amplification with primers LTR-A and (CAC)7T Kalendar PNAS 97: 6603 (2000)

14. BARE-1 copy number at different locations in canyon in = integrase encoded within BARE-1 North-Low (& South-Low): - less stress - lower BARE-1 copy number “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.” Kalendar PNAS 97: 6603 (2000)