Transposons in Eukaryotes (Part A): Structures, Mechanisms and Applications

Abstract

Transposable elements (TEs) are DNA (deoxyribonucleic acid) segments which can mobilise from their original location and (re)insert into new positions in the genome. TEs occur in almost all eukaryotic genomes. Half of the human genome consists of TEs and large plant genomes are composed of more than 80% TEs. Prevailing among these are the retrotransposons (class I elements) that propagate through an RNA (ribonucleic acid) intermediate. DNA transposons, or class II elements, move as DNA by a cut‐and‐paste mechanism. The vast majority of transposon insertions into genes result in gene damage. It is therefore of ‘mutual interest’ of host cells and transposons to limit transpositions to very low frequencies. Eukaryotes normally keep transposons under control by epigenetic inactivation via transcriptional and post‐transcriptional gene silencing. However, in rare cases, transposon insertions also lead to altered gene regulation or to the development of novel cellular functions, a process termed ‘domestication’ of transposons. In genetic research, TEs are used as versatile tools for insertion mutagenesis and gene isolation.

Key Concepts

  • Transposons are evolutionary old components of almost all eukaryotic genomes.
  • Transposon contents in eukaryotic genomes vary from <1% to >85%.
  • By default, all transposons in a genome are epigenetically silenced by DNA (deoxyribonucleic acid) and histone modification.
  • Environmental stress (‘genome shock’) can lead to release of epigenetic silencing and reactivation of transposons.
  • Transposon insertions into genes are almost always deleterious, but in rare cases can result in novel gene regulation patterns.
  • Very rarely transposons were ‘domesticated’ to provide novel functions to the host.
  • Transposons can be applied as genetic tools for mutagenesis and gene isolation.

Keywords: retrotransposons; DNA transposons; DDE transposase family; genome evolution; gene silencing; transposon tagging; insertion mutagenesis

Figure 1. The two major classes of eukaryotic transposons. (a) Retrotransposons (also termed class I elements) transpose by a ‘copy‐and‐paste’ mechanism. In the first mobilisation step, they are transcribed into RNA (TS), followed by reverse transcription into cDNA (RT) and integration of the cDNA (INT) into novel positions in the genome (blue and green lines). (b) DNA transposons (class II elements) move by a ‘cut‐and‐paste’ mechanism. During transposition (TP), the transposon ends associate with a target DNA (blue line), are excised from the donor site and reintegrated into the target DNA, followed by ligation of the donor‐site ends. In a simple transposition reaction, the two donor site ends are sealed by nonhomologous end joining (NHEJ) which typically results in a ‘footprint’ (FP) with small mutations at the fusion site. More rarely, the excision site can also be repaired by homology‐dependent gap repair using the homologous chromosome in diploid cells as template, resulting in replicative transposition. Helitrons, a subclass of DNA transposons, move by a different transposition mechanism that always results in replicative transposition.
Figure 2. Architecture and transposition mechanism model of LTR retrotransposons. These elements are terminated at both ends by identical long terminal repeats (LTRs) which consist of the functionally distinct U3, R and U5 segments. The U3 region contains a promoter (bent arrow). Autonomous LTR retrotransposons encode several proteins that are either individually expressed or post‐translationally processed from a polyprotein by a protease (prot). RT‐RNaseH: reverse transcriptase with a C‐terminal ribonuclease H domain. endo: integrase. gag: structural polyprotein precursor including nucleocapsid protein(s). env: envelope protein in retroviruses, nonfunctional envelope protein in endogenous retroviruses (ERVs). PBS: primer binding site. PPT: polypurine tract. Open triangles: short target‐site duplications flanking LTR retrotransposon insertions. 1: In the first step of mobilisation, LTR retrotransposons are transcribed by RNA Pol II into a polyadenylated genomic RNA that starts at the 5′ end of the R region in the 5′ LTR and terminates behind the R region in the 3′ LTR. 2: Reverse transcription is initiated by binding of a cognate tRNA to the PBS, where its 3′‐end serves as a primer for first‐strand cDNA synthesis by RT towards the 5′ end of the genomic RNA. The reverse transcribed RNA 5′ end is degraded by RNaseH. 3: The cDNA is translocated to the RNA 3′ end (1st template switch) where it hybridises to the R region. 4: first‐strand cDNA synthesis is continued by RT towards the PBS at the RNA 5′ end. 5: Consecutively with reverse transcription, the RNA is degraded by RNaseH, except for the RNaseH‐resistant PPT region. 6: The PPT primes second‐strand cDNA synthesis towards the PBS‐complementary region of the tRNA. 7: The residual PPT and tRNA are degraded and the second‐strand cDNA is translocated to the 3′ end of the first‐strand cDNA where it hybridises to the PBS region. 8: cDNA synthesis is completed. 9: The integrase (endo) catalyses the insertion of the double‐stranded cDNA into the genome and the generation of short target‐site duplications.
Figure 3. Architecture of non‐LTR retrotransposons and the ‘target‐primed reverse transcription’ (TPRT) transposition model. (a) Human LINE‐1 (or L1) is the prototype of long interspersed nucleotide elements that are widespread in eukaryotic genomes. L1 consists of a 5′ untranslated region (5′‐UTR) with a promoter (bent arrow) and a 3′‐UTR with a polyadenylation signal (AATAAA) followed by a variable number of A nucleotides (…(A)n). L1 has two internal open reading frames. ORF2 encodes the reverse transcriptase (RT) and an endonuclease (EN); ORF1 encodes a RNA‐binding protein (RBP) that is essential for transposition. Open triangles: short target‐site duplications that vary in length are flanking non‐LTR retrotransposon insertions. 1: In the first step of mobilisation, LINE elements are full‐length transcribed by RNA Pol II into a genomic RNA. 2: The LINE‐encoded endonuclease (EN) generates a single‐strand nick in its consensus recognition site 5′‐TTTTA‐3′. The question mark indicates that the details of second‐stand cleavage are unknown yet. 3: The LINE RNA anneals with its polyA‐tail to the TTTT‐motif that serves as primer for reverse transcription (dashed line), while the LINE RNA is concomitantly degraded during first‐strand cDNA synthesis by RNaseH. 4: The 3′‐end of the LINE cDNA is matched to the EN‐generated 3′‐overhang followed by second‐strand synthesis and gap filling. The mechanistic details of this process are unknown. 5: The new copy of the LINE element is flanked by target‐site duplications variable in length. (b) SINEs (short interspersed nucleotide elements) are nonautonomous retrotransposons. Mobilisation of SINEs depends on the LINE‐encoded ORF1 and ORF2 proteins. Like LINEs, they are flanked by target‐site duplications variable in length. Complete Alu elements are ∼300 bp in length, contain at the 5′‐end a RNA Pol III promoter (A–B) and terminate in a polyA‐tail. Their body consists of two signal recognition particle 7SL‐RNA‐derived sequences (L/R‐7SL) that are separated by a A‐rich sequence (A‐r). SVA (SINE–VNTR–Alu) elements consist of a variable‐length CCCTCT repeat, an inverted Alu‐like sequence, a variable number of tandem repeats (VNTR), a sequence derived from human endogenous retrovirus HERV‐K10 (SINE‐R), a polyadenylation signal (AATAAA) and a polyA end. Whether SVA transcription is initiated from an internal promoter or they are read‐through transcribed from upstream promoters is unknown (bent arrows). (c) Processed pseudogenes originate from spliced messenger RNAs (E1, E2, E3: exons 1, 2, 3) that were accidentally reverse transcribed into cDNA and integrated into the genome by LINE proteins. They lack a promoter and are typically not mobile.
Figure 4. Architecture and transposition mode of TIR DNA transposons. (a) Autonomous DNA transposons encode their own transposase protein (TPase), which binds to the terminal‐inverted repeats (IR) and catalyses the DNA cleavage and joining reactions during transposition. Some DNA transposons require additional proteins for transposition (ORF2) that may be expressed from separate promoters or derive from alternatively spliced RNAs. DNA transposon ends may contain additional, subterminal binding sites for the transposase or other proteins (orange dots), which are required for or modulate transposition. DNA transposons generate target‐site duplications (TSD) of defined length upon insertion. (b) Nonautonomous DNA transposons lack a functional transposase gene but can be mobilised in trans by transposase. (c) Transposition model for the hAT superfamily of DNA transposons. 1: TPase synapses both transposon ends and a target DNA into a nucleoprotein complex (‘transpososome’). The nucleotides that flank the transposon 5′‐ends, G and T, are arbitrarily chosen to illustrate the excision site repair process. TPase initiates transposition by single‐strand cleavage one nucleotide distal from the transposon 5′‐ends (red arrows). 2: The exposed 3′‐OH groups attack the phosphodiester bonds at the transposon 3′‐ends (red arrows) resulting in release of the transposon and formation of covalently closed DNA hairpins in the flanking DNA. 3: The 3′‐hydroxyl groups of the transposon perform a nucleophilic attack on the target DNA, resulting in strand transfer of the transposon to the target DNA. The architecture of the transpososome determines an offset of the attacked phosphodiester bonds of the target DNA strands (8 bp for hAT transposons). 4–5: The excision site hairpins are opened by single‐strand cleavage at variable distance 3′ from the nucleotide at the centre of the hairpin (blue arrows). This cleavage is catalysed by a cellular DNA repair endonuclease. 6: The target DNA at the transposon insertion site is repaired by fill‐in synthesis and removal of the single nucleotide overhang at the transposon 5′‐end. The excision site is repaired by nonhomologous end joining (NHEJ). During that reaction, the single‐strand overhangs generated by hairpin opening may either be exonucleolytically degraded (red triangle) or filled in (green dashed arrow) upon association with the opposite 3′‐end. 7: After reinsertion, the transposon is flanked by newly generated target‐site duplications (TSD). The repaired excision site carries an individual ‘transposon footprint’. If one or both overhangs after hairpin opening were fill‐in repaired, the footprints will contain palindromic duplications centring around the complementary nucleotide of the one that previously flanked the respective transposon end.
Figure 5. Architecture of Helitrons and the ‘rolling circle’ transposition model. (a) Helitrons are characterised by a conserved TC at the 5′‐end, followed by a 22 nt AT‐rich region and a GC‐rich palindromic sequence (‘GC‐rich hairpin’) upstream of the conserved CTRR sequence (most frequently CTAG) at the 3′‐end. Helitrons insert into AT target sites. (b) Helitrons are proposed to transpose by a ‘rolling circle’‐like mechanism. 1: Transposition is initiated by single‐strand nicking at the 5′‐end of the Helitron and one strand of the target DNA. 2: Strand transfer of the Helitron 5′‐end to the target DNA is catalysed. 3: A helicase unwinds the Helitron DNA followed by ‘rolling circle’ replication of the Helitron. When replication reaches the Helitron 3′‐end, the displaced Helitron strand is cleaved. 4: The replicated Helitron strand is ligated to the 5′‐end of the donor DNA strand and the liberated 3′‐end of the displaced Helitron strand is transferred to the free 5′‐end of the target DNA. 5: The single‐strand Helitron insertion is stabilised by mitotic DNA replication, resulting in one daughter cell carrying the novel Helitron insertion.
Figure 6. Hypothetical model for the DNA strand‐transfer reaction by transposases and integrases. In this ‘two‐metal ion model’, it is proposed that the acidic DDE amino acid triad plays a key role in the catalysis of the nucleophilic attack of a 3′‐hydroxyl group to a phosphodiester bond in the target DNA by coordinating two divalent cations which are essential for the reaction. The incoming transposon end is shown in blue and the target DNA in brown colour. (a) An electron pair from the hydroxyl group at the transposon 3′‐end displaces the bond of the phosphorus atom with the 3′‐end of the target DNA (red arrows). (b) The products of this transesterification reaction are the transposon strand transferred into the target DNA strand and a liberated target DNA 3′‐OH‐end.
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Wang, Zhenxing, and Kunze, Reinhard(Jun 2015) Transposons in Eukaryotes (Part A): Structures, Mechanisms and Applications. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0026264]