Transposable Elements

Question 1

What are transposable elements?

Answer 1

‘Mobile DNA’ capable of copying themselves around the genome

As they move around they change/block expression from nearby genes

Question 2

What are the 2 kinds of TE?

Answer 2

DNA transposons, ‘cut & paste’ DNA (Class II)

Retrotransposons, ‘copy & paste’ through RNA (Class I)

Question 3

Why is there so much TE in the genome and why does it vary?

Answer 3

Different transposition rates
Acquisition of new TEs
Efficiency of removing TEs

Question 4

Why are there TEs?

Answer 4

TEs are ‘selfish’ genes, a sequence that can copy itself around genome will increase in frequency until stopped - if you copy from 1 parent will be passed to over half offspring
Even if causes harm, as long as reduction in fitness less than benefit of spreading
(Arguably most abundant parasites)

Question 5

How do ‘copy & paste’ RNA retrotransposons work?

Answer 5

Copy themselves via RNA intermediate
Transcribed as RNA, reverse transcriptase expressed
Reverse transcribed to another part of the genome

Question 6

How do ‘cut & paste’ DNA transposons work?

Answer 6

2 methods:

1. Repair using sister chromatid
   - excised during replication
   - repair excision using homologous part of genome, which is the same as the transposable element
   - leaves 3 copies
2. Move ahead of replication
   - jumps ahead of replication fork so host will produce 2 copies

Question 7

What consequences can TE insertions have?

Answer 7

Reading frame/promoter of protein can be broken and expression stopped
TEs can carry functional coding sequences which can be broken & expression stopped
Can lead to targeting of heterochromatin which can spread to nearby sequences

Question 8

What are the consequences of HK2?

Answer 8

HK2 is retro-transposing element
Polymorphic insertion polymorphism in intron 17 of gene in 5% alleles
Inserting HK2 alters expression of gene that’s associated with dopaminergic signalling
Carrying allele doubles chance of being chronic injection drug user

Question 9

How can TEs cause rearrangements?

Answer 9

Movement of TEs mean there are near-identical sequences everywhere
Allows ectopic recombination which can delete and duplicate large chunks of genome
Likelihood increases exponentially as number TEs increase

Question 10

When should TEs jump?

Answer 10

Transposition bad for host
No reason to transpose in somatic cells, could harm host with no benefit to TE so could have self regulation
In germline may be bad for host but increases frequency of TE in next generation, expect defence and escape

Question 11

What do we know about ‘cut & paste’ DNA transposons (Class II)?

Answer 11

Generally 1-5k kbp long & encode single protein - a transposase that mediates excision & insertion
Inverted terminal repeats at each end, required for transposition
When inserted flanked by short direct repeats generated by target site duplications

Question 12

What are P-elements?

Answer 12

‘Cut & paste’ transposon - contain single transposase gene with 3 intron & 4 exons
Cause hybrid disgenesis: if maternal line carries P-element but paternal doesn’t offspring fertile, opposite then offspring infertile

Question 13

What is the mechanism of P-element action?

Answer 13

DNA cleavage of both strands by transposase encoded by TE
Excised transposon finds DNA with appropriate target site
Makes staggered cut & inserts
DNA copied using target site as template & ligated - results in target site duplication

Question 14

How does Mariner transposition happen?

Answer 14

Cut 2 nucleotides inside transposon at right end
Dimerisation of transposase and second nick 2 nucleotides in on other stand at 5’ end of TE
Then second cut is made on both uncut strand at end of TE
The hydroxyl ends mediate integration at TA site

Question 15

What are some recently discovered DNA transposons?

Answer 15

Helitrons, Polintons, Cryptons

Question 16

How do Helitrons transpose?

Answer 16

Nick TE plus strand at the LTS and replicate
Leading strand synthesis reconstitutes donor TE
Lagging strand synthesis results in dsDNA circle
Circle may serve as transposon donor
Can start replicating in rolling-circle replication or insert into the genome

Question 17

What do we know about RNA retrotransposons?

Answer 17

‘Copy & paste’ elements
Encode a reverse transcriptase
2 informal subclasses: LTRs and non-LTRs incl. LINE

Question 18

What are non-LTRs?

Answer 18

LINE-like elements
Have 1 or more ORFs and express a mRNA-like product encoding reverse transcriptase, usually transcribed by host RNA pol II
Most truncated in genome at 5’ end due to incomplete transcription

Question 19

How are non-LTRs transcribed?

Answer 19

Following transcription & nuclear export, transcripts translated & proteins form ribonucleoprotein particle (RNP)
RNP imported into the nucleus & non-LTR reverse transcribes from mRNA into another location in the genome
If there’s a polyA tail, cuts near a polyT motif so polyA tail can bind - BUT depends on TE-encoded nuclease, can be sequence-specific
1. Top strand cleavage - upstream or downstream
2. Template jump from mRNA to top strand of target site
3. Plus strand synthesis using cleaved top strand as primer, either:
- completion of synthesis and fill in leads to target site duplication
- completion of synthesis and degradation of non-homologous ends leads to target site deletion

Question 20

What are LTRs?

Answer 20

Generally 5-7 kbp long, encode 2/3 ORFs
Characterised by long terminal direct repeats at each end
pol gene encodes integrase (IN), reverse transcriptase (RT), & RNAse H domains
Protease which processes primary protein product containing gag & pol can be encoded by either

Question 21

How are LTRs transcribed?

Answer 21

tRNA binds at a priming site near 5’ end
This primes the start of reverse-transcription, which proceeds through a unique sequence & then repeat to 5’ end on RNA template
Repeat sequence of new DNA can now bind 3’ end of RNA
Primes continued reverse transcription through whole TE transcript
RNA template degraded by RNAse H leaving a fragment that primes 2nd strand DNA synthesis
Synthesis moves through 3’ unique sequence, the repeat and 5’ unique sequence
This fragment primes from 5’ end
Synthesis completes in both directions
Resulting dsDNA enters nucleus with viral integrase allows 3’ OH of each strand to attack target at sites a few bps apart - short target site duplication

Question 22

What else do LTRs encode?

Answer 22

Gag protein, associates with LTR element transcripts to form virus-like particles
Env if gene present contributes to forming virus-like particles - may contribute to cross-species transmission

Question 23

How are LTRs and retroviruses similar?

Answer 23

Same genes in same order
Replicate in same way
Difference is that LTRs aren’t horizontally transmitted, at least probably not often

Question 24

What are ERVs?

Answer 24

Endogenous retroviruses

Retroviruses that cannot transmit horizontally

Question 25

What are non-autonomous elements?

Answer 25

If autonomous element suffers deletion in transposase sequence & is no longer able to transpose itself and so uses transposase of another TE as still has terminal inverted repeats
Means it can become very small - eg. MITEs

Question 26

What are Alu elements?

Answer 26

SINEs in human genome
Dependent on LINEs - non-LTRs
Sequence that’s become non-autonomous element byt gaining motifs necessary to be transposed by an autonomous element
Ancestrally derived from RNA component of SRF
Interval RNA pol III promoter thought to initiate transcription
3’ end has A-rich region required for transposition

Question 27

How do KRAB domain zinc fingers act as a defensive mechanism for TEs?

Answer 27

Kruppel-associated box zinc-finger proteins (KRAB-ZFPs) - largest family of transcription factors
Bind DNA via tandem zinc-finger domains & form stable complex with TRIM28 via KRAB domain
Recruit other chromatin-related co-repressors including inactive chromatin
~2/3 KRAB-ZFBs bind to TEs and transcriptionally repress target TES - BUT most TEs still unbound

Question 28

How does the piRNA pathway act as a defensive mechanism for TEs?

Answer 28

Supresses TEs
Long transcripts transcribed from ‘piRNA generating clusters’ which comprise neighbouring & usually non-functional TEs in heterochromatic regions

Question 29

How does piRNA maturation occur?

Answer 29

In somatic fly cells piRNA intermediates with 5’ uracil generated by nuclease from cluster transcripts
Intermediates loaded to PIwi & 3’ end by another cleavage/exonuclease forming ‘primary’ piRNAs
Following methylation enters into nucleus to mediate transcriptional silencing of TEs

Question 30

How does Ping-Pong amplification happen?

Answer 30

Aub-piRNA bind & cleaves TE transcripts
Post-transcriptional silencing
Cleavage creates 5’ end of new piRNA loaded into Ago3
Ago-3-piRNA bind & cleave cluster transcripts to generate more antisense piRNAs
Transcriptional silencing - brings heterochromatin modifiers & leads to heterochromatin formation

Question 31

How do piRNAs cause hybrid dysgenesis?

Answer 31

P-elements: offspring of paternal strain carrying element = sterile, of maternal strain carrying element = unsterile
Suggests cytoplasm in egg of mother carries piRNAs conferring immunity to P-element

Question 32

Are all TEs bad?

Answer 32

Can contribute useful things:
1. TEs are mutagenic & mutations can be good
2. Provide complex mutations that include ORFs and regulation
Eg. Doc, non-LTR retrotransposon increases expression of cytochrome P450 which confers resistance to DDT

Question 33

How are TEs ‘domesticated’ in Drosophila?

Answer 33

Drosophila telomeres have lost telomerase & multiple non-LTR elements like Het-A or TART
Only inserted at end of chromosome
May be ‘arms race’ as host evolves to keep domesticated TEs under control & they evolve to escape