1. Evolutionary studies of key components of
RNAi in yeasts and the regulation of Argonaute
proteins by the Hsp90 machinery and kinases
CELL 671/672
Yang Wang
Hobman/LaPointe Lab
January 25th, 2013
2. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S. castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
3. RNA interference (RNAi): A process triggered by double-stranded RNAs (siRNA,
miRNA, piRNA) which leads to gene silencing in a sequence-specific manner.
Ender and Meister (2010)
RNAi in mammalian cells
Q1: What is the minimum requirement for a functional RNAi system?
Q2: How are RNAi components regulated temporally and spatially?
4. Mello and Conte (2004)
Lee et al. (2004)
Model organisms to study RNAi
Seatle TILL project
. Gwilliam et al (2002)
RNAi
C. elegans A. thaliana
S. pombeD. melanogaster
Merry (2001)
S. cerevisiae
Do all budding yeast species lack RNAi?
Kay et al. (2002)
M. musculus
6. H. sapiens Argonaute 2
S. pombe Argonaute 1
S. castellii Argonaute 1
Domain architecture of Argonaute proteins
Structure of Argonaute proteins is highly
conserved across evolutionary lineages
MidPAZN
Schirle and MacRae (2012)
8. NTD
H. sapiens
Dicer
S. pombe Dicer1
S. castellii Dicer1
Non-canonical Dicer processes small RNAs
Drinnenberg et al. (2009)
PAZ
9. S. castellii Ago1 and Dcr1 silence a GFP reporter
Drinnenberg et al. (2009)
GFP
10. Reconstitution of S. castellii RNAi in S. cerevisiae
RNAi negative
S. cerevisiae
S. castellii Ago1
S. castellii Dcr1
GFP (or URA3)
Silencing constructs
RNAi positive
S. cerevisiae
Dcr1
Ago1
GFP
11. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S. castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
13. GFP flow cytometry of S. castellii RNAi in S. cerevisiae
+ScaAGO1
+ScaDCR1
+HA-ScaAGO1
+ScaDCR1-myc
+ induction
− induction
Q: What will happen if we introduce
human RNAi components?
14. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S.castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
20. Weinberg et al. (2012)
At least two different mechanisms for Dicers
21. Weinberg et al. (2012)
At least two different mechanisms for Dicers
H. sapiens
Dicer
22. Weinberg et al. (2012)
At least two different mechanisms for Dicers
23. “Mid-term” summary
1. Introduction of S. castellii Argonaute and Dicer
restores RNAi in S. cerevisiae.
2. Human Argonaute 2 and S. castellii Dicer restores RNAi
in S. cerevisiae.
3. Argonaute proteins are highly conserved and
their functions are interchangeable in S. cerevisiae.
4. Dicer proteins are divergent across evolutionary lineages.
5. The reconstitution of RNAi in S. cerevisiae provides
a platform for further research.
24. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S. castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
25. RNAi
RNAi: a master regulator of gene expression
Defend against viruses
Heterochromatin
formation
Transposon silencing
Transcriptional (RITS)
Post-transcriptional (RISC)
regulation of gene expression
Abiotic stress responses
Contributes to proliferation,
differentiation, development,
and apoptosis
Bioinformatics analyses suggest that up to 60%
of human genes may be regulated by miRNA.
Regulation of RNAi?
26. Dicer Argonauteds siRNA/miRNA
siRNA/miRNA
precursors
mRNA cleavage/
translational repression
The regulation of RNAi pathway
1. The conformational change of Argonaute to accommodate
dsRNA loading?
A bi-lobed molecule with a central cleft for dsRNA binding
2. Kinases/phosphatases regulate Argonaute and/or Dicer?
?
28. Taipale et al. (2010)
Amino-terminal domain (NTD)
Middle domain(MD)
Carboxy-terminal domain (CTD)
The ATPase cycle of Hsp90 and co-chaperones
29. Hsp90 is required for pre-RISC formation in D. melanogaster
S2 cell
lysates
No GA
+ GA
Densitometric analysis
of the UV cross-link
0, 10, 30, 60, 90 min
0, 10, 30, 60, 90 min
Incubate with siRNAs
end-labeled with 32P
GA: geldanamycin
Hsp90 inhibitor
30. Hsp90 is required for pre-RISC formation in D. melanogaster
Miyoshi et al. (2010)
Q: Is the regulation of Argonaute by
Hsp90 conserved in budding yeasts?
31. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S.castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
33. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S.castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
34. The phosphorylation sites of human Ago2
Ser387 Tyr529
Tyr529 completes a small RNA 5′-end-binding pocket with
Lys 533, Gln 545 and Lys 570 (Meister 2009)
H. sapiens Argonaute 2
Ser253 Ser798
Thr303 Thr307 Tyr393
Phosphorylation regulates Argonaute activity
35. RNAi positive: very low level of forward and reverse pericentromeric transcripts
RNAi negative: the forward and reverse pericentromeric transcripts accumulate rapidly
Djupedal and Ekwall (2009)
Assay for RNAi in S. pombe
1. Single copy of Ago1 and Dcr1
2. RNAi regulates heterochromatin formation
39. Three scenarios
Kinase(s) X Pericentromere
transcription
RNAi apparatus
(ago1, dcr1)
Y
1. Kinase(s) X directly regulate Ago1 or Dcr1.
2. Kinase(s) X indirectly regulate Ago1 or Dcr1 through factor Y.
3. Kinase(s) X independently regulate pericentromeic transcription.
40. Analysis of interaction network in S. pombe
ago1
dcr1
rdp1
atf1
pcr1
kinase
sty1
phosphatase
pyp1
wee1
byr1
pmk1
pka1
hsp90
cdc37
Cdc2
Cdc25
gsk3 lsk1Cdc14
41. Project 1. Evolutionary studies of RNAi in S. cerevisiae
Background
Reconstitution of S. castellii RNAi in S. cerevisiae
Human Argonaute 2 and S.castellii Dicer restores RNAi in S. cerevisiae
Project 2. The regulation of Argonautes by Hsp90 and kinases
Background
Hsp90 regulates reconstituted RNAi in S. cerevisiae
Kinase screening in S. pombe for regulators of RNAi
Conclusions and future work
Acknowledgements
Outline
42. 1. S. castellii and human Argonautes both restore RNAi in
S. cerevisiae with S. castellii Dicer.
2. S. castellii and human Argonautes are subject to
S. cerevisiae Hsp90 regulation.
3. Argonaute proteins are highly conserved while Dicer
proteins are divergent across evolutionary lineages.
4. Kinases/phosphatases regulate key RNAi components.
Conclusions
43. Future work
1. Optimizing co-IP conditions to verify the physical interaction
between yeast Hsp90 and S. castellii and human Argonautes.
2. Localization patterns of Argonaute and Dicer proteins in S. cerevisiae
3. S. pombe phosphatase screening. Complementation studies of
identified kinase/phosphatase.
4. Investigate phosphorylation sites of S. pombe Ago1. Identify
homologs in S. cerevisiae and human cells.
5. Build an interaction network including RNAi apparatus, Hsp90 and
co-chaperones, kinases, and phosphatases.
44. Thanks!
Supervisors Committee member
Dr. Tom Hobman Dr. Richard Rachubinski
Dr. Paul LaPointe
LaPointe Lab
Rebecca Mercier
Katie Horvat
Annemarie Wolmarans
Aleksandra Janowicz
Allen Fu
Heather Armstrong
BaoChan Mai
Hobman Lab
Zaikun Xu
Dr. Jungsook Park
Dr. Justin Pare
Joaquin Lopez
Eileen Reklow
Virus group members
50. Wow, it hurts……
Incompatibility between RNAi and killer in S. cerevisae
Killer system:
(1) An endemic viral system cytoplasmatically inherited as a double-stranded RNA (dsRNA).
(2) Encodes a protein toxin that kills nearby cells while conferring immunity to cells making the toxin.
61. RIM11(YMR139W)
MRK1(YDL079C)
Interacts with cdc14 which is thought to play a role in the initiation and
completion of mitosis. Involved in the positive regulation of mis12.
wee1 Protein kinase that acts both on serines and on tyrosines. It acts as a
dosage-dependent negative regulator of entry into mitosis (G2 to M
transition). Phosphorylates and inhibits cdc2.
SWE1(YJL187C)
gsk3
byr1 Serine/threonine protein kinase involved in conjugation and sporulation. It is
thought that it is phosphorylated by the byr2 protein kinase and that it can
phosphorylate the spk1 kinase.
pka1
TPK1(YJL164C)
TPK2(YPL203W)
Activated by cAMP. Belongs to the protein kinase superfamily. AGC
Ser/Thr protein kinase family. cAMP subfamily. Contains 1 AGC-kinase C-
terminal domain. Contains 1 protein kinase domain.
62. SLT2(YHR030C)
Activated by tyrosine and threonine phosphorylation by skh1/pek1.
Regulates cell integrity and functions coordinately with the protein kinase C
pathway (pck1 and pck2). Involved the regulation of wall architecture, cell
shape, cytokinesis in exponential and stationary phase, and metabolism of
ions.
ckb1 Plays a complex role in regulating the basal catalytic activity of the alpha
subunit .CKB1(YGL019W)
pmk1
lsk1 P-TEFb-associated cyclin-dependent protein kinase Lsk1
CTK1(YKL139W)
Editor's Notes
Quickly go through the canonical RNAi pathways in eukaryotes
Foucus (Focus) on Dicer and Argonaute
RNAi: Fast response to stress
Importance:
(At least 30% of human genes are subject to regulation by RNAi)
Mechanism ---- Tool ------ Therapy
Arabidopsis thaliana, express multiple dicer homologs which specifically act against different viruses. In some cases, plant genomes also express endogenous siRNAs in response to bacterial infection.
Among animals, Drosophila, shows antiviral innate immunity against pathogens such as Drosophila X virus, through RNAi mechanism.
RNAi mechanism blocks the action of transposons (mobile elements in the genome) and maintains the genome stability.
Facilitating Gene-knockdown, Application in functional genomics, Medical application, Biotechnological application
Lower the bar to screen RNaseIII proteins. Found non-canonical Dicer
Isolating 18- to 30-nucleotide(nt) RNAs from S. castellii, K. polysporus, and C. albicans and preparing sequencing libraries
representing the subset of small RNAs with 5′-monophosphates and 3-hydroxyls (10), which are the chemical features of Dicer products.
The small RNAs of S. castellii and K. polysporus were most enriched in 23-RNAs beginning with U, and those of C. albicans were most enriched
in 22-nt beginning with A(adenine) or U (uracil) (Fig. 1B). These biases were reminiscent of those observed for Argonaute-bound guide RNAs of animals, plants,
and other fungi (11–13).
Rnase III domains work in pairs to nick both strands of an small RNA duplex
S.castellii Dcr1 may act as a homodimer
The restored RNAi pathway silences both introduced marker genes
and endogenous transposons.
I really don’t like the numbers here. Bullets or just the words would be better. I agree with Tom about the font; Calibri or Arial would be better. It is future work, not future works.
Your heading doesn’t make any sense. Why is SC+2%Galactose inside the box in the top right but nowhere else? I also would suggest making the figure this way – seeing the following duplicate.
Rule out the possibility that RNAi negative pairs are due to the lost or non-expression of key components
A pair of Dcr1DC dimers (green and yellow) modeled with dsRNA (sphere model). The anticipated 23 nt siRNA product is highlighted (cyan). The dsRBD is not
shown for clarity.
A pair of Dcr1DC dimers (green and yellow) modeled with dsRNA (sphere model). The anticipated 23 nt siRNA product is highlighted (cyan). The dsRBD is not
shown for clarity.
A pair of Dcr1DC dimers (green and yellow) modeled with dsRNA (sphere model). The anticipated 23 nt siRNA product is highlighted (cyan). The dsRBD is not
shown for clarity.
Processing bodies(PBs) and stress granules(SGs) are the two main type of ribonucleoprotein complexes with Argonautes are associated.
Processing bodies(PBs) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation.
amino‑terminal domain (NTD), middle domain(MD) and carboxy‑terminal domain (CTD)
Schematic of the maturation of the progesterone receptor mediated by the cooperation between heat shock
protein 90 (HSP90), HSP70 and co‑chaperones. The progesterone receptor binds HSP40 and HSP70, which then binds the
HSP90–HOP (HSC70 and HSP90‑organizing protein; also known as p60 and STI1) complex, leading to delivery of the
client. Finally, the co‑chaperone p23 (also known as PTGES3 and as Sba1 in yeast) and ATP bind the complex, promoting
client maturation and complex dissociation. The role of cyclophilin 40 (CYP40; also known as PPID) is not well understood
but it regulates the activity of client proteins in the final stages of the chaperone cycle.
Make sure you says something about Hsp90 and what GA is and what it does…this kind of comes out of nowhere.
Hsp90 is required for pre-RISC formation.
siRNA, were individually incubated with S2 cell lysates in the presence or absence of geldanamycin (geld).
Ago2 association with the siRNA duplexes was severely affected by Hsp90 inhibition, although Dicer2 association
with the duplexes was barely affected. Dicer2 association with the duplexes was slightly affected.
siRNAs end-labeled with 32P for visualization.
Densitometric analysis of the cross-link is shown by percentages. The numbers indicated on the gel image represent siRNA incubation time in lysates before 10 min
UV cross-link.
Hsp90 is required for pre-RISC formation.
siRNA, were individually incubated with S2 cell lysates in the presence or absence of geldanamycin (geld).
Ago2 association with the siRNA duplexes was severely affected by Hsp90 inhibition, although Dicer2 association
with the duplexes was barely affected. Dicer2 association with the duplexes was slightly affected.
siRNAs end-labeled with 32P for visualization.
Densitometric analysis of the cross-link is shown by percentages. The numbers indicated on the gel image represent siRNA incubation time in lysates before 10 min
UV cross-link.
Since RNAi modulates gene expression on a global level, it is critical to understand how this process is regulated
Wee1 modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases.
Atf1 and pcr1 are transcriptional factors
5-fluoro-orotic acid medium
5-FOA can be used to select for mutant cells that fails to utilize orotic acid as the source of pyrimidine ring,
Wild type cells convert 5-FOA to 5-FUMP(5-fluoro-uridine monophosphate), which is quite toxic to the cell. The reaction is catalyzed by the products of the yeast genes URA5 and URA3.
Phylogenetic tree of representative fungal species, indicating the presence of dsRNA killer viruses (blue shading), the presence of RNAi (green shading), and inferred loss of RNAi (red x).
Schematic of the 2D domain structure of human Dicer with crystal structures homologous to each module.
Segmented map of human Dicer with crystal structures of homologous domains docked.
(b)Model for pre-miRNA recognition. A pre-miRNA hairpin is modeled into the proposed binding channel of Dicer, with the stem-loop fit in the RNA-binding cleft of the helicase.
Domain architectures of representative RNase III proteins. The N-terminal domain (NTD) unique to budding-yeast RNase III enzymes is indicated.
Crystal structure of a Dcr1DC dimer at 2.3A ° resolution, showing a pair of NTDs (red and pink), a pair of RNase III domains (blue and silver), and a single dsRBD1
(green). The other dsRBD1 had poor density and is not shown. Disordered loops are shown as dotted lines, and VL-1 and VL-2 are labeled.
I really don’t like the numbers here. Bullets or just the words would be better. I agree with Tom about the font; Calibri or Arial would be better. It is future work, not future works.
Heading is awkward.
Schematic doesn’t make sense.
Really emphasize that the hit is the same as Joachin’s...very strong evidence.
