Systems Biology approaches to investigate the biogenesis and function of pirnas in Insects. Attilio Pane ICB/UFRJ

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1 Systems Biology approaches to investigate the biogenesis and function of pirnas in Insects Attilio Pane ICB/UFRJ

2 Regeneration Cancer and stem cell biology Genome stability Gene regulation Recognition of the self Genome stability Gene regulation

3 RNAi mechanisms in Drosophila pirna clusters Dcr1 Dcr2 Aubergine (Aub) Argonaute3 (Ago3) mirnas (21-22nt) mirnp sirnas (21-22nt) RISC pirnas (23-30nt) Piwi Translational repression RNA degradation Silencing of Transposable Elements

4 Drosophila ovary Anterior Posterior Oocyte Anterior Posterior Germarium Germline Somatic Follicle Cells

5 Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity D A P V

6 Transposons can cause: - Insertional mutagenesis - Double-strand DNA breaks - Recombination - Telomere fusion Therefore They are a major source of genomic instability

7 Quase medade do genoma humano sao elementos transponiveis!

8 Doencas geneticas humanas causadas por transposons Selvam Ayarpadikannan and Heui-Soo Kim, 2014

9 Transposon density in Drosophila Approximately 30% of the Drosophila genome is made up of transposon and repetitive elements. 2002

10 Ago3 Aub pirnas (23-30nt) 5 3 antisense C pirna precursor Transposon transcript Chr2 Rhi Del Cuff Chr3 N Piwi Piwi centromere pirna cluster Active transposon

Nuage Aub e Ago3 são proteínas Argonauta que

organito citoplasmatico que envolve o nucleo da

11 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. Fruitfly egg chamber (nuage) Nucleos das celulas nutridoras

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

Os elementos transponiveis causam danos no genoma quando se mobilizam A Via

estabilidade genomica A atividade dos pirnas e das proteinas Piwi

Conforme, tanto em invertebratos (Drosophila) quanto em vertebratos

esterilidade nos individuos adultos Wildtype mutante cuff Caspase3 α-spec

13 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 Wildtype mutante cuff Caspase3 α-spec DNA Caspase3 Na ausencia de pirnas, os transposons se mobilizam e Causam danos nas Celulas Germinativa que sao Eliminadas por apoptose.

14 Cuff accumulates in nuclear foci Cuff-EGFP Actin DNA Cuff-EGFP Pane et al. (2011) The Embo J

15 The RDC Complex R= Rhino (Rhi), Heterochromatin Protein 1 variant D= Deadlock (Del), no conserved domains C= Cutoff (Cuff), Rai1-like transcription co-factor DNA Cuff Rhi Del Klattenhoff et al., (2009) Pane et al., (2011) Mohn et al. (2014) Pritykin et al. (2017)

16 Major pirna clusters in Drosophila (dual-strand) Chr2L Chr2R Cluster 1/42AB Transposon remnants 250Kbp Cuff Del Rhi Transposon orientation Transcription Sense and Antisense pirnas

17 Analysis of the pirna population in the cuff mutant ovary Cuff Del Rhi pirna clusters Aub Ago3 pirnas Piwi Silencing of Transposable Elements

Normalized reads Normalized reads -2e+05-1e+05 0e+00 1e+05 2e+05-3e+05-2e+05-1e+05 0e+00 1e+05 The pirna population is depleted in cuff mutant ovaries e-sirnas (21-22nt) pirnas

18 Normalized reads Normalized reads -2e+05-1e+05 0e+00 1e+05 2e+05-3e+05-2e+05-1e+05 0e+00 1e+05 The pirna population is depleted in cuff mutant ovaries e-sirnas (21-22nt) pirnas (23-29nt) wt Small RNA libraries (Next Generation Sequencing) Length (nt) e-sirnas (21-22nt) pirnas (23-29nt) cuff Length (nt)

19 pirna density along cluster 1/42AB Chr 2L Chr 2R Cluster 1/42AB Small RNA library Sequencing 250Kbp wt cuff + strand - strand Uniquely mapping pirnas

20 Mutations in cuff impair tranposon silencing Cuff Del Rhi pirna clusters Aub Ago3 pirnas Piwi Silencing of Transposable Elements

21 The pirna pathway is active early during germline development Reporter puas gfp Zenv Transposon sequence Stem cell region Reporter α-spec DNA wt cuff rhi aub Reporter Pane et al., (2011) The Embo J..

22 cuff mutant Cuff activity is required for transposon silencing RNAseq Wild type 40 transposon families are upregulated in the cuff mutant ovary

23 Ago3 Aub pirnas (23-30nt) 5 3 antisense C pirna precursor Transposon transcript Chr2 Rhi Del Cuff Chr3 N Piwi Piwi centromere pirna cluster Active transposon

24 Mature eggs display Anterior-Posterior and Dorsal-Ventral polarity D A P V

25 Mature eggs laid by cuff mutant females display severe DV polarity defects Category 1 Category 2 Category 3 Category 4 wild type weakly ventralized moderately ventralized strongly ventralized

26 Establishment of the Dorsal-Ventral axis Gurken = TGF-α-like molecule D D A A DNA Actin Gurken A D

27 Functional pirna pathway (wt) pirna pathway mutants (cuff) Transposon silencing Transposon deregulation DNA damage (double strand breaks) Meiotic Checkpoint (Chk2 kinase)? Gene expression

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