Lecture 15/16 - Transposable Elements Flashcards
What is the most common type of transposable element mutation?
Deletion or insertion
When do transposable elements mutations take place?
Anytime during development
What are transposable elements?
Mobile DNA which jump in and out of genomes blocking or changing gene expression
What are the 2 types of transposable elements?
DNA transposomes and Retrotranspones (RNA)
Do all organisms have the same amount of TEs?
No
Is the proportion of the genome made up by TE variable?
Yes
Why is the proportion of the genome made up of TEs variable?
Due to transposition rates,
Acquisition rates of new TE
Efficiency of selection in removing TEs
Why is there lots of TE’s?
Any sequence that can copy itself around the genome will increase in frequency until something stops it
Do TE’s get passed on more commonly than other alleles?
Yes because there is more copies
Can harmful TE’s be safe passed on?
Yes
Retrotransposons - copy and paste
How does this work?
Copy themselves using RNA and then reverse transcribe into another part of the genome
How do cuts and paste “transposons” work?
You haven’t increased numbers therefore you need to cut yourself out and paste yourself somewhere else. Then the DNA will be repaired and will therefore copy the TE
What is the other mechanisms for cut and paste dna transposons which is more hypothetical?
If the TE is cut out and goes somewhere else then that will eventually be replicated when the replication fork goes through again
What are some consequences if TE’s?
They break reading frames and therefore stop the original proteins expression
They have functional coding sequences themselves which impacts neighbouring genes
They can also target heterochromatin and stop the expression of lots of genes
What are some subtler consequences of TE’s? Human example - HK2 is a retro-transposing element?
It alters the relative amount of the expression of 2 transcripts in the body - doesn’t take them out entirely.
If you have this it double your chances of being a chronic injection drug user
Are TE’s insertions big or small?
Small
What disease was the TE’s insertion of LINE first recognised in?
Haemophilia
Why is having TE’s dangerous even when they aren’t inserting themselves?
The amount of TE’s you have clumped together might trick the body into thinking it’s a site of recombination (ectopic recombination)
Why is ectopic recombination bad?
Could lead to large deletions
Do recombinations between TE’s be only in the same chromosome? What happens if it is in different chromosomes?
No it causes duplicated regions in one chromosomes and a deleted region in the other chromosomes
What is the chance of recombinations equal to?
The square of the number
Do genes which could get rid of TE’s spread rapidly?
Yes until all the TEs are fixed
What diseases is caused by TE jump?
Cancers
If a TE jumps in a brain cell say will it be inherited by the offspring?
No
When wouldn’t a TE jump?
If it is not in a cell that will be passed to offspring e.g if it’s in an egg cell then yes if it’s in muscle tissue cell then no
When could TE’s be useful?
They are mutagenic and some mutations could useful. They provide complex mutations which include ORFs and regulation
Example of good TE’s?
Making drosophila more resistant to insecticides
What does it mean when a TE becomes domesticated? Example?
This means the TE’s are used in a way needed by the body. For example in drosophila the telomeres can go missing and a TE will continuously jump in and replace these
How do you identify TE’s in a genome?
You use a read.
You fragment a genome and compare it to the non fragmented reference genome.
You then compare this to a genome with TE’s and see what’s different
In what circumstances is using reads good? And why is it bad in the other when finding TE’s?
TE poor areas is better than TE rich areas as it’s hard to then tell the TE’s sort of there’s lots of them
When trying to identify mutations you can also use a mutation accumulation line. What is this?
This is when you mate flies and mate their offspring and their offspring etc, as it allows you to generate new mutations and then compare it to your parental flies to see what’s new
Different populations can have different numbers of TE’s why?
Due to specific elements that are present in each flies, e.g some have more LTR’s than others
Why is it hard to see where new TE’s instert?
Some are rare or dangerous
How do we get passed the problems of detecting TE’s for example how rare they are?
You activate TEs by removing suppression mechanism and compare TE insertions in the original generation with offspring
What is the most common area for TEs to insert?
Promotors - especially transcription start sites
Highly expressed genes and euchromatin suggesting access is an important factor
Where are surviving TE’s inserted?
Introns
Are some TE’s specific to target sites
Yes this includes short signal motifs
Why would TE’s target low impact regions?
It reduces cost and increases fitness
What are the class II (DNA cut and paste transposons) most studied groups?
Mariner and P elements
What is involved in a transposon?
1kb-5kb Long and encode a single protein which is usually a transposase
What does a transposase do?
Mediates excision and insertion
What are the inverted terminal repeats at both ends for?
Required for transposition
What is the transposase flanked by when inserted into the genome?
Shirt direct repeats generated by target site duplications
How does class II DNA cut and paste works?
The TE is cut out and inserted into another genome which is slightly staggered. The repair of this staggered cut it what causes the direct repeats which are a target site duplication
What is mariner transposition? And how is it copy and pasted? Step 1
A transposase being expressed by the sequence binds to the terminal inverted repeat and the sequence is cut.
What is mariner transposition? And how is it copy and pasted? Step 2
Then either a dimerised transposase binds the other to create a dimerised transposase with a loop of dna between it. There is then a second cut to leave a free loop of dna and the dimerised transposase
What is mariner transposition? And how is it copy and pasted? Step 3
Dimerised transposase cuts both ends and this becomes a free loop of DNA and free OH groups which mediate attack in target site
What is mariner transposition? And how is it copy and pasted? Step 4
Transposes recognised target site and cuts either side of a TA and this is inserted leaving short regions which need to be repaired (e.g the lagging area).
Are TE’s of different variations cut in similar ways?
Yes but in a different order
How do we recognise a new TE?
Look for repetitive sequence
Look for recognisable sequences such as terminal inserted repeats and transposase.
Look for any cross species transfer
Helitrons are a family of TE’s and don’t work in the same way as p-elements and mariner sequences. So what is the difference?
When inserted into a genome this family had one strand cleaved and replication if the strand left in genome makes the double stranded dna.
The free copies form circles and this can happen over and over.
These circles undergo rolling circle replication making lots of replication which can be left or can be reinserted into the genome.
Another class of TE’s are polintons and have their own polymerase what is it called?
DNA polymerase D
Polintons also have a integrate. What is this called?
DDE-like integrase
We don’t know how polintons transpose - what is the theory?
They create a circle of DNA which cleaves and the polymerase replicates DNA which can then be reintegrated
Lecture 1 and 2
What the two subclasses of retrotransposons?
LTR - long terminal repeat
Non-LTRs include LINE (long interspersed nuclear elements, Penelope-like elements and DIRS
What did LINE’s use for?
Reverse transcriptase
What are LINES transcribed by?
RNA Pol II
Where are LINES truncated about?
5’ end due ti reverse transcription
How do LINES transpose?
After transcription they are translated and proteins form a ribonucleoprotein particle (RNP)
These are put back into genome and synthesised
What happens after the RNP is made?
It is put back into the target site and the lower strand of the target site is cleaved. The mRNA binds.
After mRNA bind to the target site what happens?
The OH on the DNA the mRNA has bound to can start synthesis of the mRNA.
In LINE transposon the new DNA has been synthesised from the OH group and mRNA. What is one of the two products and how is this made?
RNA
Top strand cleaves and there is a template jump from the mRNA to the top strand of the target site. Plus strand synthesis then takes place using the top strand as a primer
In the RNA product of LINE transposon what two things happens after plus strand synthesis?
Completion of synthesis leads to target site duplication
Completion of synthesis and degradation of non-homologous ends leads to target site deletion
If the top strand cleaves downstream in LINE transposon what one of the 2 things that happen after plus strand synthesis happens here?
Target site duplication
If the top strand cleaves upstream in LINE transposon what one of the 2 things that happen after plus strand synthesis happens here?
Target site deletion
LTR - what does the pol gene in LTRs ENCODE?
Integrate, reverse transcriptase, protease and RNAse H domains
How does LTR’s Retrotranspose?
RNA exported with TE’s to cytoplasm and then when expressed a tRNA binds at a 5’ binding site.
How does LTR’s Retrotranspose - after tRNA has bound?
Starts reverse transcription which proceeds through a unique sequence and then a repeat to the 5’ end on the RNA template
How does LTR’s Retrotranspose - After reverse transcription?
The repeat sequences of the new DNA binds to the 3’ end of the RNA and the primers reverse-transcribe through all of the TE.
How does LTR’s Retrotranspose - After all of the TE has been reversed transcribed you need to make a DNA strand. how is this done?
RNA template degraded by RNAse H leaving fragments that primes the second strand of DNA synthesis.
How does LTR’s Retrotranspose - What happens when making the new DNA strand after RNAse synthesise DNA?
Synthesis moves through the 3’ unique synthesis sequence, the repeat and the 5’ unique sequence
How does LTR’s Retrotranspose - what happens after synthesis had moved along the DNA and through all the unique sequences?
Fragment then primes from the 5’ end and synthesis is completed in both direction
How does LTR’s Retrotranspose - what happens after synthesis is completed?
DsDNA enters nucleus with a viral integrate which allows the 3’ OH to attack the target sites a few base pairs apart leading to short target site duplication
LTR replication might also happen via a circular intermediate how does this happen?
The same way as LTR replication in non circular intermediaries apart from instead of the second strand transfer jumping to the other end of the sequence a circle is formed. this circle is mediated by hijacking an alternative end joining repair mechanism. the polymerase then replicated forming a circle. This circle is then broken and inserts into host genome.
What does the GAG protein associate with the LTR elements to do?
Forms virus like particles
What does the env gene do to the LTR elements when present?
Forms virus like particles and may contribute to cross-species transmission
Where do LTR’s replicate before going into the genome?
Virus like particles
What is the difference between LTR’s replicating in virus like particles and a retrovirus?
Retroviruses form full viral particles which exist outside of cells but so do gypsy (a LTR) so the only difference is a retrovirus can be transmitted
How is a LTR retroelement such as gypsy and HIV similar?
Contain the same genes in the same order and they replicate in the same way
What is the difference between LTR retro elements such as gypsy and HIV?
LTR’s are NOT horizontally transmitted
What are the relatives of true retroviruses that have lost their horizontal transmission?
Endogenous retrovirus (ERVs)
Where are retroviruses and ERVs found?
Only in vertebrates
Koala retrovirus is one boundary between a TE and a retrovirus - what does this cause?
Lymphomas and leukaemia caused by koala gammaretrovirus
The koala retrovirus is in two forms - what are they both and what do they do?
KoRV-B - horizontally transmitted retrovirus
KoRV-A - endogenous retrovirus (inherited in genome) retaining its ability to transmit horizontally - this would be a retroelements however it can still be infectious
Polintons could also be a virus cross over - how?
They encode their own DNA polymerase which is similar to viruses.
Polintons also encode capsid proteins which are viral particles that could be horizontally transmitted like a virus
Non-autonomous elements - Hyper-parasitism - What would happen if a mutation took out the transposase from the TE?
The other transposable elements making their own transposase will recognise the terminal inverted repeats letting it transpose.
This therefore is a parasite of the other transposable elements
What do a mutated transposase TE make when they use another TE’s transposase?
Miniature inverted-repeat transposable elements (MITE’s).
Why can SINE sequences become non-autonomous
Gaining motifs necessary to be transposed by an autonomous element
Alu elements - SINEs in the human genome - where does it come from?
RNA component of the signals recognition particle ribonucleoprotein complex
Alu elements - SINEs in the human genome - what transcribes these?
RNA polymerase III
Alu elements - SINEs in the human genome - what is it dependant on to retrotransposition?
LINE-1 ORF2p
Alu elements - SINEs in the human genome - are most of these active?
No
Immunity against TE’s - what is the Krabbe domians and zinc fingers?
Transcription factors that bind via tandem zinc-finger domains and form stable complex with TRIM28 via KRAB domain
Immunity against TE’s - what does TRIM28 do?
Recruit chromatin related corepressors inducing inactive chromatin in the region of the TE therefore stopping them transcribing genes
Immunity against TE’s - what does the piRNA pathway do?
Suppresses transposable elements
Immunity against TE’s - how does the piRNA pathway stop TE’s?
Transcripts are transcribed from piRNA generating closets which neighbour TEs in heterochromatin
Immunity against TE’s - PiRNA maturation - how does this happen?
Cluster transcript is cleaved so everything starts with a U. Is then loaded into Piwi and then cleaved. this forms piRNA’s.
Following thisv2-O methylation by Hen-1 the mature PiRNA-piwi complex enters nucleus and mediates transcriptional silencing of TE”s.
Immunity against TE’s - piRNA pathway ping pong amplification - how does this work?
Sun-piRNA (cluster piRNA) bind and cleave TE transcripts resulting in post transcriptional silencing
What does cleavage of the Aub pi-RNA do?
Creates a 5’ end of a new piRNA which is loaded onto ago3
What does Ago3 do?
Ago3-piRNA binds and cleaves cluster transcripts to make more amends piRNA’s
What helps maturation of aub pi rna
Bubbler or zucchini cleavage
What happens when zucc products associate with lieu
They enter the nucleus to mediate transcriptional silencing of te
What do the mature oiRNAs bound to piwi do?
Recruit repressive chromatin marks in the nucleus
In drop Julia what do pirna-piwi bind to
Nascent TE transcripts
What do the nascent TE transcripts do?
Induced co transcriptional repression through recruitment of general silencing machinery components - repressive H3K9me3
What happens when H3K9me3 recruits HP1
Heterochromatin formation
What is required for transcriptional silencing and blocks H3K9me3 spread?
Maelstrom
