Lecture 20

Question 1

Transposable elements cause problems:

Answer 1

• They can mutagenise protein coding genes by inserting between a coding sequence
• They can increase in number, so require host cell resources
• They threaten the integrity of the genome as they can increase in copy number

Question 2

Class 1: Retrotransposons:

Answer 2

• Copy and paste mechanism
• long terminal repeat sequences, pol (reverse transcriptase), gag (integrase
• RNA molecule produced by pol, and makes a DNA copy which integrates back into the host genome using gag
• increase in genome

Question 3

Class 1b: nonviral-like retrotransposons

Answer 3

• Copy and paste
• Differ in that they lack long terminal repeats, but they still have an RNA indermediate
• LINEs and SINEs

Question 4

Class 2: DNA transposons:

Answer 4

• Cut and paste mechanism

- Just hop around the genome, and only increase in number if they move at the right time of DNA replication

Question 5

How can you stop transposable elements causing a problem?

Answer 5

• Target the RNA transcripts

- Wrap them up in chromatin

Question 6

Distribution of TEs in the human genome:

Answer 6

• Retro-elements (RNA intermediate elements) cause the most problems for humans

Question 7

IF you were a transposable element, in what tissue would you want to be most active?

Answer 7

• In the germ line cells so that you contribute genetic material to the next generation
• Can also be active in other cells, but germ line cells are the most logical

Question 8

Indentifyication of fly PIWI mutants:

Answer 8

• Mutants displaying defects in gametogenesis and are sterile

Question 9

PIWI (P-element induced wimpy testis):

Answer 9

• Spermatogeneseis defects arise from precocious activation of retrotransposons in the male germ line
• Oogenesis defects arise from a loss of stem cells as a result of DNA damage signaling

Question 10

Mammalian PIWIs and gonad development:

Answer 10

• 3 PIWIs in humans
• Arrest of gametogenesis and complete sterility in males
• Activation of LINE and retrotransposons in Mili/Miwi2 mutants

Question 11

PIWI is a member of the Argonaut family:

Answer 11

• PIWI’s are only found in animals

- AGO’s are found in animals, plants and fungi

Question 12

PIWI’s are only active in the germline

Answer 12

• This is clearly a pathway specific for the germline

Question 13

piRNA’s

Answer 13

• PIWI interacting RNAs
• Incredibly diverse (1.5 million different piRNAs in flies)
• Mostly derived from retrotransposons, transposons and repetitive elements

Question 14

piRNA clusters in the genome:

Answer 14

• 80% of piRNS’s map to specific genomic loci

- usually antisense to the TE mRNA, giving a strong bias for uridine at the 5’ end

Question 15

piRNA’s role

Answer 15

• Preventing transposable element mobilisation in the germline
• Prevent DNA damage
• Prevent telomere loss (composed of retrotransposon repeats)

Question 16

piRNA modes of action:

Answer 16

• Target retrotransposon/transposon RNA for endonucleolytic cleavage (slicing) in cytoplasm (post-transcriptional gene silencing)
• DNA methylation/chromatin modification of transposons in genome (transcriptional gene silencing)

Question 17

piRNA biogenesis:

Answer 17

• a piRNA cluster is made up of sense and antisense RNA
• This is chopped up to be loaded into PIWI.
• The processing differs in the nucleus to the cytoplasm

Question 18

Does piRNA need dicer in the cytoplasm?

Answer 18

• No, instead it uses exonucleases

Question 19

Loading the piRNA into RISC produces active piRISC. Where do these go?

Answer 19

• Some go into the nucleus, where transcriptional silencing occurs with the help of Pol2
• Some go into the cytoplasm where post-transcriptional silencing occurs, using TE transcript cleavage

Question 20

What is ping-pong amplification?

Answer 20

• The way that lots of piwi’s can be generated

- TE sense and antisense piRNAs are associated with different subclasses of PIWIs and have a 10nt overlap

Question 21

Ping pong cycle:

Answer 21

• Binding of the retrotransposon transcript with a PIWI type 1 results in slicing
• PIWI type 2 binds at the 10A sequence
• It goes into the cytoplasm for processing
• a piRISC complex forms
• It can now basepair with the pre-RNA transcript
• The sliced transcript is then recognised by the first class of PIWIs
• A secondary piRNA derived from the pre-cursor transcript binds again.

Question 22

What does the ping-pong amplification loop achieve?

Answer 22

• Massive amplification of piRNAs
• Silences TE through cleave of their transcripts
• Leads to the amplification of useful piRNAs (they have to be able to target transposons)

Question 23

Defence against transposable elements, the main problems:

Answer 23

• There is massive diversity within TE
• TE can evolve rapidly
• TE can jump species barriers

Question 24

Hybrid dysgenesis:

Answer 24

• Crossing lab bread flies with WT flies results in F1 sterility
• This is due to TE present in the wild flies become active in the captive flies
• The captive population cannot control the TE
• If you study the F1 over time they can regain fertility, as the p-elements jump into a piRNA cluster and then piRNAs can be generated for it

Question 25

piRNA clusters are programmable loci in a way. What does this mean?

Answer 25

- When a TE jumps into a piRNA cluster, a piRNA can be produced for it - Until then, there may be no piRNA for that specific piRNA