W8L2 Discovery and fuction of piRNAs

Question 1

Piwi mutants

Answer 1

Piwi (P-element induced wimpy testis) mutants have defects in gametogenesis and are sterile
Piwi involved in processes underlying germline development – but may also be involved in a pathway involved in hybrid dysgenesis

Question 2

evidence suggesting that Piwi is involved in regulating TE activity

Answer 2

TE activity is elevated in the germline of Piwi mutants – mainly affects retrotransposons but some DNA transposons

Question 3

Hybrid dysgenesis

Answer 3

• Crossing a WT female with a mutant L male lead to infertile offspring
  -Crossing mutant L female with male lead to fertile offspring
  -This is due to maternal factor present

Question 4

Piwi genes encode Ago-like protein

Answer 4

Two group of Argonaute protein: Ago (animals, plant, fungus) and Piwi Ago (animal germ line specific)
Piwi/Aub/Ago3 expressed in BOTH male/female germline and closely associated cells
Aub also accumulates in pole plasm of the oocyte before fertilization

Question 5

Small RNAs associated with Piwi proteins

Answer 5

Characterize RNAs associated with fly Ago proteins following crosslinking + immunopreciptation (CLIP) procedure:
Non-Piwi Ago (act as control): Ago1 around 21-22nt
Piwi-Ago: Piwi, Ago3, Aub around 24-30nt

Question 6

diversity Result of small RNA associated with PIWI protein test

Answer 6

Incredibly diverse - 1.5 million piRNAs in fly oocytes
Most derived from retrotransposons, transposons and repetitive elements
Called Piwi-interacting RNAs: piRNAs

Question 7

Mammalian Piwis and piRNAs

Answer 7

§ Three Piwis in mice (Mili/Miwi/Miwi2) – mainly expressed in male germline
§ Arrest of spermatogenesis and complete sterility in male mili/miwi2 mice mutants associated with activation of LINE and retrotransposons

Question 8

Two type of mammalian piwis

Answer 8

Pre-pachytene piRNAs are derived from TE (retrotransposons)
Pachytene piRNAs lack sequences matching TEs

Question 9

Pi-RNA cluster

Answer 9

§ 80% piRNAs map to specific genomic loci – bidirectional vs unidirectional fly – pericentromeric/sub-telomeric regions
mouse – small clusters of complex DNA in euchromatic regions
§ Cluster length varies (2-200kb) – 40-4000 piRNAs present
§ Variable piRNA density within cluster
§ Enriched for transposon and other repeats – but inactive remnants
§ Associated with H3K9me3 marks – required for transcription

Question 10

Production of piRNAs

Answer 10

pi-cluster in genome – sense/antisense remnants of TE/repetitive sequences
* piRNA precursor is large, single-stranded and lacks a foldback loop
* No 5’ Cap, polyA tail and no promoter
* Dicer not involved in production of piRNAs
* Processing of piRNA precursor into mature piRNAs occurs in perinuclear RNA granules called nuage or Yb bodies – U-enriched at position 1 and antisense to TE sequences
-processed by Piwi-Argo and exonucleases in nuage, loaded into Aub and Piwi (24-30nt antisense)

Question 11

piRNAs – mode of action

Answer 11

1. Target retrotransposon/transposon mRNA for endonucleolytic cleavage (slicing) in nuage (post-transcriptional gene silencing) - Aub
2. Chromatin modification of TEs in nucleus (transcriptional gene silencing) - Piwi

Question 12

Amplification of piRNAs

Answer 12

Amplification cycle increases the abundance of piRNAs in the germline that target active TEs and repetitive elements – ping-pong
§ piRNAs associated with Aub-RISC are antisense to TE sequences and have U at position 1
§ piRNAs associated with Ago3-RISC are mainly identical to sense TE sequences and have a bias for A at position 10
§ 10nt overlap between piRNAs associated with Aub-RISC and Ago3-RISC

Question 13

piRNA biogenesis – the ping-pong cycle

Answer 13

-Aub-containing piRISC binds to complementary TE mRNA
-Slicing inactivates the TE mRNA, Creates a recognition site for Ago3
-Ago3 binds to the cleaved TE transcrip
-Ago3-bound TE transcript is processed into a sense piRNA
-Ago3-containing piRISC binds to the piRNA precursor
-Aub binds to the cleaved piRNA precursor
-Aub-bound piRNA precursor is processed into an antisense piRNA

Question 14

How does hybrid dysgeneisis work

Answer 14

As all RNA present during the early stages of embryogenesis is maternally-derived – look at the piRNAs in embryo vs maternal parent

Question 15

Characterising maternally inherited piRNAs

Answer 15

piRNA profile of F1 embryo and female ovary are identical
Aub-associated piRNAs to I elements are deposited into the developing oocyte of I females

Question 16

Maternally inherited piRNAs prime defence against TEs

Answer 16

Following fertilisation maternally-inherited Aub-piRNAs:
Trigger the ping-pong cycle in the nuage of pole cells of the embryo
Enter the nuclei of pole cells and install H3K9me3 marks on the pi-RNA clusters – enhance transcription of piRNA clusters

Question 17

Key issues facing genomic defense mechanism:

Answer 17

1. Diversity of TE with little similarity at the primary sequence level
2. TE evolve rapidly
3. TE can jump species barriers e.g. D melanogaster (1900’s), D. simulans (1990)

Question 18

pi-RNA clusters as TE traps

Answer 18

pi-RNA clusters function as TE traps - programmable silencing loci
Acquire new content through innate mobility of TEs
When TEs land in a pi-RNA cluster immunity to that TE is immediately established –adaptive immunity

Question 19

Test of the TE trap model

Answer 19

1. If TE depression occurs – piRNA clusters play an important role in TE control
2. If TE repression is maintained – piRNA clusters may not be the principal regulators of TE activity

Question 20

piRNA clusters are not required for TE control

Answer 20

Delete three most highly expressed germline pi-RNA clusters
No change in fertility
Massive decrease in piRNAs
No change in TE expression

Question 21

TE control is mediated in cis theory

Answer 21

For many TEs, primary source for piRNA production is provided by dispersed full-length TE insertions rather than the germline-expressed piRNA clusters
TE regulation may be achieved in cis by piRNAs derived from dispersed elements

Question 22

Invasion dynamics of P-element in D. erecta

Answer 22

Introduction of P element into a naïve genome
Maintain flies for 50 generations:
genome sequencing, RNA-seq and small RNA-seqs
Repeated 3 time:
R1/R4 – P element numbers remain constant from gen 20 onwards (siRNA increasing in abundance, piRNA low, afterward piRNA increasing in abundant)
R2 - P element numbers increases exponentially from gen 20 onwards()

Question 23

Crank up model

Answer 23

1. TE insertion in the genome triggers endo-siRNA production
2. Cleaved TE RNA substrates for piRNA processing (v. small number)
3. Piwi-associated piRNAs enter the nucleus and convert a TE insertion into a piRNA-producing locus
4. Endogenously produced piRNAs amplified by the ping-pong cycle
5. Control of TEs established