Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos...
Transcript of Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos...
![Page 1: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/1.jpg)
Attilio Pane
ICB/UFRJ
Systems Biology approaches to investigate the biogenesis and function of piRNAs in Insects
![Page 2: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/2.jpg)
Regeneration
Genome stability Gene regulation
Genome stability Gene regulation
Cancer and stem cell biology
Recognition of the “self”
![Page 3: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/3.jpg)
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
![Page 4: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/4.jpg)
Anterior Posterior
Germline Somatic Follicle Cells
Oocyte
Germarium
Drosophila ovary
Anterior Posterior
![Page 5: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/5.jpg)
A P
D
V
Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity
![Page 6: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/6.jpg)
Transposons can cause:
- Insertional mutagenesis
- Double-strand DNA breaks
- Recombination
- Telomere fusion
Therefore
They are a major source of genomic instability
![Page 7: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/7.jpg)
Quase medade do genoma humano sao elementos transponiveis!
![Page 8: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/8.jpg)
Doencas geneticas humanas causadas por transposons
Selvam Ayarpadikannan and Heui-Soo Kim, 2014
![Page 9: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/9.jpg)
Transposon density in Drosophila
2002
Approximately 30% of the Drosophila genome is made up of transposon and repetitive elements.
![Page 10: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/10.jpg)
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)
![Page 11: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/11.jpg)
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
![Page 12: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/12.jpg)
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
![Page 13: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/13.jpg)
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.
![Page 14: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/14.jpg)
Cuff-EGFP Actin DNA
Cuff-EGFP
Cuff accumulates in nuclear foci
Pane et al. (2011) The Embo J
![Page 15: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/15.jpg)
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)
![Page 16: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/16.jpg)
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
![Page 17: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/17.jpg)
Analysis of the piRNA population in the cuff mutant ovary
Silencing of Transposable Elements
piRNAs
Piwi
Aub
Ago3
piRNA clusters Rhi
Cuff Del
![Page 18: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/18.jpg)
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)
![Page 19: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/19.jpg)
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
![Page 20: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/20.jpg)
Silencing of Transposable Elements
piRNAs
Piwi
Aub
Ago3
piRNA clusters
Rhi
Cuff Del
Mutations in cuff impair tranposon silencing
![Page 21: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/21.jpg)
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
![Page 22: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/22.jpg)
Cuff activity is required for transposon silencing
RNAseq
˜40 transposon families are upregulated in the cuff mutant ovary
cuff
mu
tan
t
Wild type
![Page 23: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/23.jpg)
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)
![Page 24: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/24.jpg)
A P
D
V
Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity
![Page 25: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/25.jpg)
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
![Page 26: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/26.jpg)
Establishment of the Dorsal-Ventral axis
D
A
Gurken = TGF-α-like molecule
DNA
Actin
Gurken
D
A
D A
![Page 27: Systems Biology approaches to investigate the biogenesis ... Atilio.pdf · Os elementos transponiveis causam danos no genoma quando se mobilizam A Via dos piRNAs silencia os elementos](https://reader033.fdocuments.us/reader033/viewer/2022041418/5e1d08bc18b5435b32499598/html5/thumbnails/27.jpg)
Functional piRNA pathway (wt) piRNA pathway mutants (cuff)
Transposon silencing Transposon deregulation
DNA damage (double strand breaks)
Meiotic Checkpoint (Chk2 kinase)
Gene expression
?