Transposible elements Viruses and viroids

1. Transposible elementsViruses and viroids

2. Transposons, TE • = mobile geneticelements • sequencesof DNA thatcanmovearound to differentpositionswithinthe genome of a single cell (transposition), • cause mutationsandchromosomalrearrangements • identified in ProkaryotesandallEukaryotes: • (withexceptionofparasiticPlasmodium falciparum) • animals 3-45%, fungi 2-20%, plants 10-80% • in plants: • - thousandsoffamilies, form majority ofrepetitive DNA

3. Basic classification of TE • DNA transposons - „CUT and PASTE“ (rarely „COPY and PASTE“) typically: transposasecleavesout, inserts to newsite • Retrotransposons - „COPY and PASTE“ typically: - reverse transcriptase - DNA copiesfrom TE transcripts - integrase - insertion

4. Basic classification of TE - self-sufficiency • Autonomous elements – encode genes necessary for transposition/ replication • Non-autonomous – derivatives of autonomous elements – lost of genes for transposition/replication – keep sequences necessary for transposition (can be mobilized by related autonomous elements!)

5. Discovery of transposons Barbara McClintock (1902-1992) Nobel prize in Physiology and Medicine 1983 Mobile genetic elements in maize 1940-1950

6. Discovery of TE • study of chromosomal breakage • increased frequency in certain site • (= marker „dissociation“ Ds) • location of Ds was unstable after • crossing with some lines • (= line carrying „activator“ Ac)

7. Discovery of TE • in one location – Ds insertion was connected with loss of purple pigment of endosperm • - after crossing with activator line pigment synthesis was recovered in some cells

8. Barbara McClintock (1902-1992) 1951: formulated basic context of epigenetics: "[T]he progeny of two (such) sister cells are not alike with respect to the types of gene alteration that will occur. Differential mitoses also produce the alterations that allow particular genes to be reactive. Other genes, although present, may remain inactive. This inactivity or suppression is considered to occur because the genes are ‘covered' by other nongenic chromatin materials. Gene activity may be possible only when a physical change in this covering material allows the reactive components of the gene to be ‘exposed' and thus capable of functioning."

9. Classification of TE • Class: - replicationwith/without RNA intermediate • DNA transposons • Retrotransposons • Subclass: - mechanismofreplication (DNA transposones) • Order: - basic structuralfeatures • Superfamily: - similarityofsequences Nature Rev. Genet., 2007

10. Basic TE subclasses /replication protein + helicase Lisch 2013, Nature Rev. Genet.

11. Class II: DNA transposons

12. DNA transposons - subclass I • transposition: break, religation • terminal inverted repeats recognized by transposase • (fungal TE Crypton encodes recombinase instead of transposase) • - duplication of short seq. (2-8 bp) = footprint after transposition • - clustering in genom, hundreds of copies

13. Multiplication of DNA transposons Activation during replication - how? Hemimethylated state? + break repair by homologous recombination (TE can be restored)

14. Examples of DNA transposons – subclass I: - Ac, Spm, Mu (maize), Tam (Antirrhinum), TphI (petunia), TagI (Arabidopsis) - non-autonomous: Ac/Ds, Spm/dSpm - Stowaway, Tourist >10 000 copies

15. DNA transposons – subclass 2 Helitrons – single strand breake and strand displacement - Rep/helikase-like, replication protein A-like - maize: 4 - 10 000 gene fragments mobilized by helitrons

16. Class I: Retrotransposons

17. Retrotransposons • replication through RNA intermediate (multiple offspring) • related to retroviruses • millions of copies • huge portions of genome (up to 40-80 % of genome size) • element size 1-13 kbp • Order LTR – most important in plants • LTR (long terminal repeat) - promotor, terminator, direct repeat • dubling of short target sequence • gag (nucleocapsid), • pol (protease, reverse transcriptase-RNase H, integrase)

18. Retrotransposons LTR - replication • - LTR (U3, R, U5) • - PBS • tRNA primer • skips between • templates • (direct repeat)

19. Examples of LTR retrotransposons BARE-1, barley, 12,1 kbp, >50 000 copies, transcript in leaves and callus PREM-2, maize, 9,5 kbp, >10 000 copies, transcript in microspores Tnt1, tobacco, 5,3 kbp, >100, activated after wounding, patogen attack, Ty3 – gypsy group – ancestors of Caulimoviruses, hypothetical ancestors of animal retroviruses (env-like sequence) Athila, A.t., 10,5 kbp, >10000, paracentromeric regions

20. Retrotransposons without LTR LINE (long interspersed nuclear elements) SINE (short interspersed nuclear elements) LINE - phylogenetically most original, ancestors of LTR - 5´region – promoter; 3´ region - terminator Cin4, maize, 1-6,8kbp, 50-100, various truncated forms SINE - non-autonomous – use RT of other elements - derived from products of RNA polymerase III (tRNA, 7SLRNA, (rRNA)) - < 500 nt APE – endonuklease, RH – RNase H LINE

21. Regulation of transposon activity - both endogenous and by plant cell - mostly inactive – methylated (prevents activity and also illegitime recombination (crossing over) - often developmentally regulated activation - rarely activation by environmental conditions: Tam1 (1000x at 15°C) Reme1– activation by UV light

22. Maintenancemethylationof TE Zemach et al. 2013

23. Role of TE in evolution • causing mutations = increasing variability • modulation of expression (activation, repression, developmentally- or stress- induced) – important during domestication (WHY?) • new gene evolution • genome evolution • in plants no direct profit – no genes directly increasing fitness (like resistances in bacteria) • increase in fitness by random mutation – low probability, but possible (really occuring)

24. Transposon-mediated mutagenesis • - site of insertion • - character of transposon regulatory sequences • modulation of transcription (spatial, temporal) – promoter, enhancers • transcript stability and splicing • changes in protein sequence (footprints, frame-shift) – • possible role in evolution of new genes Promotor 5´UTR exon intron exon 3´UTR terminator

25. TE affected expression of TF VvmybA1 - regulation of antokyan synthesis genes (Kobayashi et al. 2004, Science)

26. TE affected gene expression - examples Maize: – inactivationofCCT (photoperiod response) by CACTA-like element (DNA TE) insertion to promoter – allowedcultivation in temporalclimate (long-dayflowering) blockofbranching (TE enhancer OE of inhibitor) (Yang et al. 2013, PNAS) Redorange: Ruby – myb TF (regulationof antokyan genes) activated by TE insertion - coldinducedexpression in fruits (Butelli et al. 2012, Plant Cell)

27. R Ds Transposon mutagenesis • mainly in Arabidopsis • insertional mutagenesis (alternative to T-DNA mutagenesis – see later) • – easy detection of the site of insertion (x chemical mutagenesis) • DNA transposons from maize – low frequency of transposition • two-component systém (transposase/Ds) Gene for transposase (inducible expression) R Ds Line with non-autonomous element in resistance gene Selection of resistant plants = with transposed transposon Selection in next for plants without transposase gene • frequent mutagenesis of near genes – clustering (20 % in 1Mb around) • reintroduction of transposase – possibility to reverse the mutation

28. Role in gene evolution • - insertional mutagenesis (premature termination), footprints • participation in gene duplications • - directly or via recombination (TE = homologous repeats) • formation of intron-less gene copies (reverse transcription) • genes of TE origin „domesticated“ by many eucaryotic organisms • for new functions (telomerase, syncitin, ….) • new gene formation by fusions of mobilized gene fragments (helitrons)

29. Role in genome evolution • Chromosomalrearrangements • - changesoflinkagegroups • - speciation (incompatibility) • Increase in genome size(„genomic obesity“) • multiplicationof TE x homologousrecombination • canpreventorevendecrease genome size (2n cottons – 2 -3x genome sizedifferences by activerecombination; Hawkins 2009 PNAS)