Attilio Pane

ICB/UFRJ

Systems Biology approaches to investigate the biogenesis and function of piRNAs in Insects

Regeneration

Genome stability Gene regulation

Genome stability Gene regulation

Cancer and stem cell biology

Recognition of the “self”

RNAi mechanisms in Drosophila

Dcr2

RISC

Translational repression

RNA degradation

siRNAs (21-22nt)

miRNAs (21-22nt)

Dcr1

miRNP

Silencing of Transposable Elements

piRNAs (23-30nt)

Piwi

Aubergine

(Aub)

Argonaute3

(Ago3)

piRNA clusters

Anterior Posterior

Germline Somatic Follicle Cells

Oocyte

Germarium

Drosophila ovary

Anterior Posterior

A P

D

V

Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity

Transposons can cause:

- Insertional mutagenesis

- Double-strand DNA breaks

- Recombination

- Telomere fusion

Therefore

They are a major source of genomic instability

Quase medade do genoma humano sao elementos transponiveis!

Doencas geneticas humanas causadas por transposons

Selvam Ayarpadikannan and Heui-Soo Kim, 2014

Transposon density in Drosophila

2002

Approximately 30% of the Drosophila genome is made up of transposon and repetitive elements.

N

C

centromere piRNA cluster Active transposon

piRNA precursor

Chr2

Chr3

Piwi Piwi

Ago3

Aub

Transposon transcript

Rhi

Cuff

Del

3’ 5’ antisense

piRNAs (23-30nt)

Nuage

Fruitfly egg chamber (nuage)

Aub e Ago3 são proteínas Argonauta que processam os transcritos Gerados pelos clusters a formar os piRNAs maduros. Esta etapa da Biogênese acontece no NUAGE, um organito citoplasmatico que envolve o nucleo da células germinativas.

Nucleos das celulas nutridoras

Cutoff (Cuff): a protein with similarity to Yeast Rai1

- Rai1 interacts with the 5’-3’ exoribonuclease Xrn2 and elicits Transcription

Termination in yeast

- Cuff does not display the catalytic domain of Rai1

- Cuff seems to actually have a transcription anti-termination function in

Drosophila (Mohn et al., 2014)

- Mutations in cuff cause female adult sterility.

Cuff

Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos transponiveis assim garantindo A estabilidade genomica A atividade dos piRNAs e das proteinas Piwi desempenha papel fundamental Na linhagem germinativa! Conforme, tanto em invertebratos (Drosophila) quanto em vertebratos (camundongo), defeitos na producao e funcao dos piRNAs resultam em esterilidade nos individuos adultos

Caspase3 α-Spec DNA

Caspase3

Wildtype mutante cuff

Na ausencia de piRNAs, os transposons se mobilizam e Causam danos nas Celulas Germinativa que sao Eliminadas por apoptose.

Cuff-EGFP Actin DNA

Cuff-EGFP

Cuff accumulates in nuclear foci

Pane et al. (2011) The Embo J

The “RDC” Complex

R= Rhino (Rhi), Heterochromatin Protein 1 variant D= Deadlock (Del), no conserved domains C= Cutoff (Cuff), Rai1-like transcription co-factor

Rhi

Cuff Del

DNA

Klattenhoff et al., (2009) Pane et al., (2011) Mohn et al. (2014) Pritykin et al. (2017)

Chr2L Chr2R

Cluster 1/42AB

Transposon remnants

250Kbp

Major piRNA clusters in Drosophila (dual-strand)

Transposon orientation

Transcription

Sense and Antisense piRNAs

Rhi

Cuff Del

Analysis of the piRNA population in the cuff mutant ovary

Silencing of Transposable Elements

piRNAs

Piwi

Aub

Ago3

piRNA clusters Rhi

Cuff Del

20 21 22 23 24 25 26 27 28 29 30 -2e+

05

-

1e+

05

0e+

00

1

e+0

5

2

e+0

5

-3e+

05

-2e+

05

-

1e+

05

0

e+0

0

1e+

05

Length (nt)

e-siRNAs (21-22nt)

piRNAs (23-29nt)

20 21 22 23 24 25 26 27 28 29 30

e-siRNAs (21-22nt)

piRNAs (23-29nt)

Length (nt)

No

rmal

ized

rea

ds

No

rmal

ized

rea

ds

wt

cuff

The piRNA population is depleted in cuff mutant ovaries

Small RNA libraries

(Next Generation Sequencing)

piRNA density along cluster 1/42AB

Chr 2L Chr 2R

wt

Uniquely mapping piRNAs

“+” strand

“-” strand

Cluster 1/42AB

250Kbp

cuff

Small RNA library Sequencing

Silencing of Transposable Elements

piRNAs

Piwi

Aub

Ago3

piRNA clusters

Rhi

Cuff Del

Mutations in cuff impair tranposon silencing

The piRNA pathway is active early during germline development

cuff rhi aub

gfp Zenv pUAS Reporter

Pane et al., (2011) The Embo J..

Transposon sequence

wt

Reporter α-Spec DNA

Reporter

Stem cell region

Cuff activity is required for transposon silencing

RNAseq

˜40 transposon families are upregulated in the cuff mutant ovary

cuff

mu

tan

t

Wild type

N

C

centromere piRNA cluster Active transposon

piRNA precursor

Chr2

Chr3

Piwi Piwi

Ago3

Aub

Transposon transcript

Rhi

Cuff

Del

3’ 5’ antisense

piRNAs (23-30nt)

A P

D

V

Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity

Category 1 Category 2 Category 3

Category 4

wild type strongly

ventralized weakly

ventralized moderately ventralized

Mature eggs laid by cuff mutant females display severe DV polarity defects

Establishment of the Dorsal-Ventral axis

D

A

Gurken = TGF-α-like molecule

DNA

Actin

Gurken

D

A

D A

Functional piRNA pathway (wt) piRNA pathway mutants (cuff)

Transposon silencing Transposon deregulation

DNA damage (double strand breaks)

Meiotic Checkpoint (Chk2 kinase)

Gene expression

?