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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
?