![Regulation of RNA interference by Hsp90 is an evolutionarily
conserved process
Yang Wang a
, Rebecca Mercier a
, Tom C. Hobman a,b,c
, Paul LaPointe a,
⁎
a
Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Canada
b
Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Canada
c
Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Canada
a b s t r a c ta r t i c l e i n f o
Article history:
Received 12 April 2013
Received in revised form 19 June 2013
Accepted 20 June 2013
Available online 1 July 2013
Keywords:
Hsp90
RNAi
Argonaute
Dicer
Yeast
RNAi is a highly conserved mechanism in almost every eukaryote with a few exceptions including the model
organism Saccharomyces cerevisiae. A recent study showed that the introduction of the two core components
of canonical RNAi systems, Argonaute and Dicer, from another budding yeast, Saccharomyces castellii, restores
RNAi in S. cerevisiae. We report here that a functional RNAi system can be reconstituted in yeast with the in-
troduction of only S. castellii Dicer and human Argonaute2. Interestingly, whether or not TRBP2 was present,
human Dicer was unable to restore RNAi with either S. castellii or human Argonaute. Contrary to previous re-
ports, we find that human Dicer, TRBP2 and Argonaute2 are not sufficient to reconstitute RNAi in yeast when
bona fide RNAi precursors are co-expressed. We and others have previously reported that Hsp90 regulates
conformational changes in human and Drosophila Argonautes required to accommodate the loading of
dsRNA duplexes. Here we show that the activities of both human and S. castellii Argonaute are subject to
Hsp90 regulation in S. cerevisiae. In summary, our results suggest that regulation of the RNAi machinery by
Hsp90 may have evolved at the same time as ancestral RNAi.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
RNA interference (RNAi) regulates gene expression at both the
transcriptional and post-transcriptional levels in eukaryotes [1,2].
Long double-stranded RNA (dsRNA) precursors undergo one or
more processing steps to generate small interfering RNAs (siRNAs)
or microRNAs (miRNAs) that are ultimately incorporated into the
RNA-induced silencing complex (RISC). RNaseIII proteins specifically
cleave dsRNA and include multiple components of the RNAi pathway
[3]. In the miRNA biogenesis pathway, primary miRNA transcripts are
processed in the nucleus by the class 2 RNaseIII proteins Drosha and
Pasha, into hairpin structures [4,5]. This step is critical to generate
2 nt 3′ overhangs at the ends of hairpins that can be recognized
by the PAZ domain of the class 3 RNaseIII protein, Dicer, which then
cleaves the double-stranded portion of the hairpin into 21–23
nucleotide dsRNAs [6–10]. Canonical Dicer proteins have been called
“molecular rulers” for their ability to cleave dsRNA a fixed distance
from the end [7]. The short dsRNAs generated by Dicer bind to a
core component of RISC called Argonaute which, depending on the
properties of the Argonaute isoform involved, cleaves or displaces
the passenger strand [11–15]. This leaves the ssRNA guide strand in
the binding cleft of Argonaute and comprises the functional core of
the RISC. It is this ssRNA-containing Argonaute complex that targets
mRNAs for cleavage or translational suppression. Despite significant
advances in our understanding of the RNAi system, the minimum re-
quirements for the pathway and how it is regulated are not fully
understood.
It is estimated that ~70% of mammalian genes are regulated by RNAi,
but not all eukaryotes possess an RNAi system [16–18]. The budding
yeast Saccharomyces cerevisiae is a powerful model organism that has
been widely used in genetic and molecular studies of eukaryotic cells,
however, it lacks any recognizable homologues of Argonaute or Dicer,
and does not carry out RNAi-mediated silencing [18]. Recent studies
show that some closely related species of budding yeasts such as
Saccharomyces castellii and Kluyveromyces polysporus, are capable of
RNAi-mediated silencing [19]. The Argonaute proteins in these fungi
share all four identified domains with human Argonaute but also have
elongated N-terminal regions, whereas S. castellii Dicer is much shorter
than canonical Dicer proteins from humans and the fission yeast
Schizosaccharomyces pombe (Fig. 1A and B). RNAi can be restored in
S. cerevisiae by introducing only Argonaute and Dicer from either
S. castellii or K. polysporus [19,20]. The reconstituted RNAi system si-
lences both exogenous reporter genes and endogenous retrotransposons.
This suggests that the last common ancestor of these budding yeasts pos-
sessed RNAi, but it was lost in some species including S. cerevisiae. The
RNAi pathway in humans is more complicated but three components,
Biochimica et Biophysica Acta 1833 (2013) 2673–2681
Abbreviations: RNAi, RNA interference; GFP, green fluorescent protein; TRBP2, Tar
(HIV)-RNA binding protein 2
⁎ Corresponding author. Tel.: +1 780 492 1804.
E-mail address: paul.lapointe@ualberta.ca (P. LaPointe).
0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbamcr.2013.06.017
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamcr](https://image.slidesharecdn.com/b5baeee8-ae73-4fa2-91a8-350b045d1def-170126034025/85/1-s2-0-S0167488913002401-main-1-320.jpg)
![Argonaute2, Dicer, and TRBP2, have been shown to be sufficient to recon-
stitute the minimal human RISC complex in vitro [21].
The important role that the RNAi system plays in regulating gene
expression necessitates that RNAi itself is subject to regulation.
Clues about the regulation of the RNAi machinery have come from
the observation that the loading of dsRNA duplexes into human and
Drosophila Argonaute is ATP-dependent [23]. Moreover, it has been
shown that the hydrolysis of ATP is catalyzed by the Hsc70/Hsp90
chaperone machinery during formation of the RISC [23–25]. We and
others have proposed that Hsp90 facilitates a conformational change
required to accommodate the RNA duplex into the binding cleft of
Argonaute. Importantly, the components of the Hsp90 system are
highly conserved in eukaryotes and many human Hsp90 client pro-
teins can be regulated by the yeast Hsp90 system [26,27]. Only
recently has the Hsp90 system been identified as a key regulator
of the RNAi system in higher eukaryotes. It is not clear however
if Hsp90 plays a role in the regulation of distantly related RNAi
systems — such as that from S. castellii.
We report here that RNAi can be reconstituted in S. cerevisiae with
S. castellii Dicer and either S. castellii Ago1 or human Argonaute2. Con-
trary to a recently published study however, we observed that human
Dicer did not support functional RNAi with either Argonaute protein,
with or without the co-factor TRBP2. Similar to RNAi in humans and
Drosophila, we found that Hsp90 plays an important regulatory role
in reconstituted RNAi systems in budding yeast; consistent with the
structural conservation of Argonaute proteins.
2. Materials and methods
2.1. Plasmids and strains
Silencing constructs were expressed from plasmids pRS403-
PGAL1-weakSC-GFP, pRS403-PGAL1-strongSC-GFP and pRS403-PGAL1-
hpSC-URA3 as previously described [19]. Untagged versions of
S. castelli Argonaute and Dicer were encoded by pRS404-PTEF-ScaAgo1
and pRS405-PTEF-ScaDcr1 (both acquired from Addgene) as previously
described [19].
We constructed the pRS406-PADH1-GFP(S65T) dual reporter plas-
mid (for uracil autotrophy and GFP fluorescence) as follows. The
SacI–KpnI restriction fragment from p414ADH1 [28] was cloned into
similarly digested pRS406 to make pRS406-PADH1. GFP(S65T) was am-
plified by polymerase chain reaction (PCR) with primers designed to
incorporate an upstream BamHI and downstream SalI sites. This PCR
fragment was cut with BamHI and SalI and cloned into similarly
digested pRS406-PADH1. The resultant reporter plasmid was used to
measure both URA3 and GFP expression in our studies.
The complete human Ago2 coding sequence was amplified by PCR
with SpeI (upstream) and SalI (downstream) sites and cloned in place
of S. castellii Ago1 in pRS404-PTEF-ScaAgo1 (digested with SpeI and
XhoI) to make pRS404-PTEF-hAgo2.
The complete human Dicer coding sequence was amplified by PCR
with SpeI (upstream) and XhoI (downstream) sites and cloned in
place of S. castellii Ago1 in pRS405-PTEF-ScaDcr1 (digested with XbaI
and XhoI) to make pRS405-PTEF-hDcr1.
pRS401-PTEF-TRBP2-natNT2 was constructed as follows. The KpnI–
SacI fragment from p414TEF [28] was inserted into the similarly cut
pRS401 to make pRS401PTEF. PCR-amplified TRBP2 (complete coding
sequence with upstream BamHI and downstream XhoI sites) was
cloned into the BamHI and XhoI sites of pRS401PTEF to make
pRS401-PTEF-TRBP2. Finally, the nourseothricin (natNT2) selectable
marker from pRS41N [29] was amplified by PCR to have an EagI site
upstream of the promoter and downstream of the terminator. This
fragment was cloned into the EagI site of pRS401-PTEF-TRBP2 to
make pRS401-PTEF-TRBP2-natNT2.
Epitope tagging of Ago and Dcr was done as follows. For human
Ago2 and S. castellii Ago1, complementary primers comprised of an
H. sapiens Dicer
(1922 aa)
S. pombe Dicer1
(1374 aa)
S. castellii Dicer1
(610 aa)
Helicase dsRBD PAZ RNase IIIa dsRBDRNase IIIb
51 602 630 722 891 1042 1276 1403 1666 1824 1849 1914
Helicase
19 517 537 628 916 1038 1083 1233
dsRBD RNase IIIa RNase IIIb
dsRBDdsRBDRNase III
119 263 274 337 537 599
NTD
H. sapiens Argonaute2
(859 aa)
S. pombeArgonaute1
(834 aa)
S.castelliiArgonaute1
(1299 aa)
235 370 517 818
213 348 499 799
585 722 937 1258
PAZ
PAZ
PAZ
N
N
N
MID
MID
MID
PIWI
PIWI
PIWI
A
B
Fig. 1. Domain architecture of Dicer and Argonaute proteins. A. Dicer proteins structure with helicase (light green), dsRNA binding (orange), PAZ (red), and RNaseIII (blue) domains
in ribbon representation. B. Argonaute protein structure with N (purple), PAZ (red), Middle (orange), and PIWI (green) domains in ribbon representation. The amino acid sequence
of Argonaute and Dicer proteins was retrieved from GenBank, and the domain boundaries predicted by SMART [41,42] and UniProt (http://www.uniprot.org/).
2674 Y. Wang et al. / Biochimica et Biophysica Acta 1833 (2013) 2673–2681](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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- 1. Regulation of RNA interference by Hsp90 is an evolutionarily conserved process Yang Wang a , Rebecca Mercier a , Tom C. Hobman a,b,c , Paul LaPointe a, ⁎ a Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Canada b Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Canada c Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Canada a b s t r a c ta r t i c l e i n f o Article history: Received 12 April 2013 Received in revised form 19 June 2013 Accepted 20 June 2013 Available online 1 July 2013 Keywords: Hsp90 RNAi Argonaute Dicer Yeast RNAi is a highly conserved mechanism in almost every eukaryote with a few exceptions including the model organism Saccharomyces cerevisiae. A recent study showed that the introduction of the two core components of canonical RNAi systems, Argonaute and Dicer, from another budding yeast, Saccharomyces castellii, restores RNAi in S. cerevisiae. We report here that a functional RNAi system can be reconstituted in yeast with the in- troduction of only S. castellii Dicer and human Argonaute2. Interestingly, whether or not TRBP2 was present, human Dicer was unable to restore RNAi with either S. castellii or human Argonaute. Contrary to previous re- ports, we find that human Dicer, TRBP2 and Argonaute2 are not sufficient to reconstitute RNAi in yeast when bona fide RNAi precursors are co-expressed. We and others have previously reported that Hsp90 regulates conformational changes in human and Drosophila Argonautes required to accommodate the loading of dsRNA duplexes. Here we show that the activities of both human and S. castellii Argonaute are subject to Hsp90 regulation in S. cerevisiae. In summary, our results suggest that regulation of the RNAi machinery by Hsp90 may have evolved at the same time as ancestral RNAi. © 2013 Elsevier B.V. All rights reserved. 1. Introduction RNA interference (RNAi) regulates gene expression at both the transcriptional and post-transcriptional levels in eukaryotes [1,2]. Long double-stranded RNA (dsRNA) precursors undergo one or more processing steps to generate small interfering RNAs (siRNAs) or microRNAs (miRNAs) that are ultimately incorporated into the RNA-induced silencing complex (RISC). RNaseIII proteins specifically cleave dsRNA and include multiple components of the RNAi pathway [3]. In the miRNA biogenesis pathway, primary miRNA transcripts are processed in the nucleus by the class 2 RNaseIII proteins Drosha and Pasha, into hairpin structures [4,5]. This step is critical to generate 2 nt 3′ overhangs at the ends of hairpins that can be recognized by the PAZ domain of the class 3 RNaseIII protein, Dicer, which then cleaves the double-stranded portion of the hairpin into 21–23 nucleotide dsRNAs [6–10]. Canonical Dicer proteins have been called “molecular rulers” for their ability to cleave dsRNA a fixed distance from the end [7]. The short dsRNAs generated by Dicer bind to a core component of RISC called Argonaute which, depending on the properties of the Argonaute isoform involved, cleaves or displaces the passenger strand [11–15]. This leaves the ssRNA guide strand in the binding cleft of Argonaute and comprises the functional core of the RISC. It is this ssRNA-containing Argonaute complex that targets mRNAs for cleavage or translational suppression. Despite significant advances in our understanding of the RNAi system, the minimum re- quirements for the pathway and how it is regulated are not fully understood. It is estimated that ~70% of mammalian genes are regulated by RNAi, but not all eukaryotes possess an RNAi system [16–18]. The budding yeast Saccharomyces cerevisiae is a powerful model organism that has been widely used in genetic and molecular studies of eukaryotic cells, however, it lacks any recognizable homologues of Argonaute or Dicer, and does not carry out RNAi-mediated silencing [18]. Recent studies show that some closely related species of budding yeasts such as Saccharomyces castellii and Kluyveromyces polysporus, are capable of RNAi-mediated silencing [19]. The Argonaute proteins in these fungi share all four identified domains with human Argonaute but also have elongated N-terminal regions, whereas S. castellii Dicer is much shorter than canonical Dicer proteins from humans and the fission yeast Schizosaccharomyces pombe (Fig. 1A and B). RNAi can be restored in S. cerevisiae by introducing only Argonaute and Dicer from either S. castellii or K. polysporus [19,20]. The reconstituted RNAi system si- lences both exogenous reporter genes and endogenous retrotransposons. This suggests that the last common ancestor of these budding yeasts pos- sessed RNAi, but it was lost in some species including S. cerevisiae. The RNAi pathway in humans is more complicated but three components, Biochimica et Biophysica Acta 1833 (2013) 2673–2681 Abbreviations: RNAi, RNA interference; GFP, green fluorescent protein; TRBP2, Tar (HIV)-RNA binding protein 2 ⁎ Corresponding author. Tel.: +1 780 492 1804. E-mail address: paul.lapointe@ualberta.ca (P. LaPointe). 0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.06.017 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr
- 2. Argonaute2, Dicer, and TRBP2, have been shown to be sufficient to recon- stitute the minimal human RISC complex in vitro [21]. The important role that the RNAi system plays in regulating gene expression necessitates that RNAi itself is subject to regulation. Clues about the regulation of the RNAi machinery have come from the observation that the loading of dsRNA duplexes into human and Drosophila Argonaute is ATP-dependent [23]. Moreover, it has been shown that the hydrolysis of ATP is catalyzed by the Hsc70/Hsp90 chaperone machinery during formation of the RISC [23–25]. We and others have proposed that Hsp90 facilitates a conformational change required to accommodate the RNA duplex into the binding cleft of Argonaute. Importantly, the components of the Hsp90 system are highly conserved in eukaryotes and many human Hsp90 client pro- teins can be regulated by the yeast Hsp90 system [26,27]. Only recently has the Hsp90 system been identified as a key regulator of the RNAi system in higher eukaryotes. It is not clear however if Hsp90 plays a role in the regulation of distantly related RNAi systems — such as that from S. castellii. We report here that RNAi can be reconstituted in S. cerevisiae with S. castellii Dicer and either S. castellii Ago1 or human Argonaute2. Con- trary to a recently published study however, we observed that human Dicer did not support functional RNAi with either Argonaute protein, with or without the co-factor TRBP2. Similar to RNAi in humans and Drosophila, we found that Hsp90 plays an important regulatory role in reconstituted RNAi systems in budding yeast; consistent with the structural conservation of Argonaute proteins. 2. Materials and methods 2.1. Plasmids and strains Silencing constructs were expressed from plasmids pRS403- PGAL1-weakSC-GFP, pRS403-PGAL1-strongSC-GFP and pRS403-PGAL1- hpSC-URA3 as previously described [19]. Untagged versions of S. castelli Argonaute and Dicer were encoded by pRS404-PTEF-ScaAgo1 and pRS405-PTEF-ScaDcr1 (both acquired from Addgene) as previously described [19]. We constructed the pRS406-PADH1-GFP(S65T) dual reporter plas- mid (for uracil autotrophy and GFP fluorescence) as follows. The SacI–KpnI restriction fragment from p414ADH1 [28] was cloned into similarly digested pRS406 to make pRS406-PADH1. GFP(S65T) was am- plified by polymerase chain reaction (PCR) with primers designed to incorporate an upstream BamHI and downstream SalI sites. This PCR fragment was cut with BamHI and SalI and cloned into similarly digested pRS406-PADH1. The resultant reporter plasmid was used to measure both URA3 and GFP expression in our studies. The complete human Ago2 coding sequence was amplified by PCR with SpeI (upstream) and SalI (downstream) sites and cloned in place of S. castellii Ago1 in pRS404-PTEF-ScaAgo1 (digested with SpeI and XhoI) to make pRS404-PTEF-hAgo2. The complete human Dicer coding sequence was amplified by PCR with SpeI (upstream) and XhoI (downstream) sites and cloned in place of S. castellii Ago1 in pRS405-PTEF-ScaDcr1 (digested with XbaI and XhoI) to make pRS405-PTEF-hDcr1. pRS401-PTEF-TRBP2-natNT2 was constructed as follows. The KpnI– SacI fragment from p414TEF [28] was inserted into the similarly cut pRS401 to make pRS401PTEF. PCR-amplified TRBP2 (complete coding sequence with upstream BamHI and downstream XhoI sites) was cloned into the BamHI and XhoI sites of pRS401PTEF to make pRS401-PTEF-TRBP2. Finally, the nourseothricin (natNT2) selectable marker from pRS41N [29] was amplified by PCR to have an EagI site upstream of the promoter and downstream of the terminator. This fragment was cloned into the EagI site of pRS401-PTEF-TRBP2 to make pRS401-PTEF-TRBP2-natNT2. Epitope tagging of Ago and Dcr was done as follows. For human Ago2 and S. castellii Ago1, complementary primers comprised of an H. sapiens Dicer (1922 aa) S. pombe Dicer1 (1374 aa) S. castellii Dicer1 (610 aa) Helicase dsRBD PAZ RNase IIIa dsRBDRNase IIIb 51 602 630 722 891 1042 1276 1403 1666 1824 1849 1914 Helicase 19 517 537 628 916 1038 1083 1233 dsRBD RNase IIIa RNase IIIb dsRBDdsRBDRNase III 119 263 274 337 537 599 NTD H. sapiens Argonaute2 (859 aa) S. pombeArgonaute1 (834 aa) S.castelliiArgonaute1 (1299 aa) 235 370 517 818 213 348 499 799 585 722 937 1258 PAZ PAZ PAZ N N N MID MID MID PIWI PIWI PIWI A B Fig. 1. Domain architecture of Dicer and Argonaute proteins. A. Dicer proteins structure with helicase (light green), dsRNA binding (orange), PAZ (red), and RNaseIII (blue) domains in ribbon representation. B. Argonaute protein structure with N (purple), PAZ (red), Middle (orange), and PIWI (green) domains in ribbon representation. The amino acid sequence of Argonaute and Dicer proteins was retrieved from GenBank, and the domain boundaries predicted by SMART [41,42] and UniProt (http://www.uniprot.org/). 2674 Y. Wang et al. / Biochimica et Biophysica Acta 1833 (2013) 2673–2681
- 3. ATG codon and the HA coding sequence was annealed to have over- hangs compatible with SpeI. This small double stranded fragment was cloned into the SpeI site in pRS404-PTEF-hAgo2 and pRS404- PTEF-ScaAgo1 and clones with the appropriate orientation were iden- tified by sequencing. The resultant plasmids were called pRS404- PTEF-HA-hAgo2 and pRS404-PTEF-HA-ScaAgo1. For human Dcr1, the C terminal domain of hDcr1 was amplified by PCR with a sense primer complementary to the sequence near an internal SacII site (5′-gagagaccgcggcagcactccccgggggtcctg-3′) and an antisense primer complementary to the hDcr1 C-terminus and harboring the myc coding sequence and a XhoI site (5′-gagactcgagctatcacagatcttcttca gaaataagtttttgttcttgggaacctgagg-3′). This fragment was cloned into the SacII and XhoI sites of pRS405-PTEF-hDcr1 to make pRS405- PTEF-hDcr1-myc. For S. castellii Dcr1, the C terminal domain of ScaDcr1 was amplified by PCR with a sense primer complementary to the sequence near an internal NheI site (5′-gagagagctagcaacatccccagt ctcagtc-3′) and an antisense primer complementary to the ScaDcr1 C-terminus and harboring the myc coding sequence and a XhoI site (5′-gagactcgagctatcacagatcttcttcagaaataagtttttgttccagattgttgc-3′). This fragment was cloned into the NheI and XhoI sites of pRS405- PTEF-ScaDcr1 to make pRS405-PTEF-ScaDcr1-myc. All our strains were constructed from W303a (MATa ura3-1 leu2-3, 112, trp1-1, his3-11, 15, ade2-1, can1-100). Table 1 summarizes the genotypes of all strains used. Briefly, each strain was constructed by integration of different combinations of pRS403, pRS404, pRS405 and pRS406 vectors harboring cloned elements expressed under the control of TEF1 or ADH1 promoters. 2.2. Flow cytometry analysis of GFP expression Analysis of GFP fluorescence was carried out as previously de- scribed [19,21]. Briefly, each strain was inoculated in synthetic com- plete media with 2% raffinose and grown overnight. Fresh cultures were then seeded from the overnight cultures (500 uL to 5 mL fresh media) and cells were grown to log-phase with either 2% glucose (non-inducing) or 2% galactose (inducing). 0.2 OD600 units of cells were harvested by centrifugation at 960 ×g for 2 min in a micro- centrifuge tube. The cell pellet was washed two times with 1 mL Table 1 Yeast strains used in this study. Strain Genotype Reference W303a MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 [22] YW1 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS406-PADH1-GFP(S65T) This study YW2 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW3 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-ScaAgo1 pRS405-PTEF-ScaDcr1 This study YW4 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-ScaAgo1 pRS405-PTEF-ScaDcr1 pRS406-PADH1-GFP(S65T) This study YW5 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-ScaAgo1 pRS405-PTEF-ScaDcr1 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW6 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-ScaDcr1-myc This study YW7 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) This study YW8 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW9 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-ScaDcr1-myc This study YW10 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) This study YW11 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW12 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc This study YW13 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc pRS406-PADH1-GFP(S65T) This study YW14 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW15 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc This study YW16 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS406-PADH1-GFP(S65T) This study YW17 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW18 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 This study YW19 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) This study YW20 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW21 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 This study YW22 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) This study YW23 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-SC-URA3 This study YW24 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-ScaAgo1 pRS405-PTEF-ScaDcr1 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-weakSC-GFP This study YW25 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-ScaAgo1 pRS405-PTEF-ScaDcr1 pRS406-PADH1-GFP(S65T) pRS403- PGAL1-strongSC-GFP This study YW26 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-weakSC-GFP This study YW27 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-ScaAgo1 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-strongSC-GFP This study YW28 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-weakSC-GFP This study YW29 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-ScaDcr1-myc pRS406-PADH1-GFP(S65T) pRS403-PGAL1-strongSC-GFP This study YW30 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-weakSC-GFP This study YW31 MATa leu2-3,112 trp1-1 can1-100 URA3-1 ade2-1 his3-11,15 pRS404-PTEF-HA-hAgo2 pRS405-PTEF-hDcr1-myc pRS401-PTEF-TRBP2-natNT2 pRS406-PADH1-GFP(S65T) pRS403-PGAL1-strongSC-GFP This study 2675Y. 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- 4. PBS before final resuspension in PBS for analysis. Cells were analyzed using the 488 nm laser on a FACSCalibur (BD Biosciences). Data were processed with CellQuest Pro (BD Biosciences) and FlowJo (Tree Star). 2.3. Growth assays Each strain was grown in synthetic complete media with 2% raffi- nose overnight. Cells were diluted to OD600 of 1.0, and 1:10 serial dilu- tions were spotted onto the appropriate plates (SC, SC-ura, or 5-FOA; containing 2% glucose or galactose) and grown at 30 °C for 3 days. 2.4. Lysate generation and western blotting Cell lysates were generated as previously described [30]. Protein lysates were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Myc-tagged proteins were detected with mouse monoclonal antibody 9E10 (Millipore) [31]. HA-tagged proteins were detected with rat monoclonal antibody 3F10 (Roche). TRBP2 was detected with mouse monoclonal antibody 1D9 (AbCam). Rabbit an- tiserum raised against yeast β-actin was a kind gift from Gary Eitzen (University of Alberta) [30]. Human Ago2 was detected with a rabbit polyclonal antibody raised against the PAZ domain of human Ago2. Human Dicer1 was detected with mouse monoclonal antibody N167/7 (Millipore). 3. Results 3.1. Argonaute and Dicer from S. castellii reconstitute RNAi in S. cerevisiae Previous studies showed that introduction of S. castellii Argonaute (ScaAGO1) and Dicer (ScaDCR1) restores RNAi in S. cerevisiae [19]. To confirm this result, we integrated plasmids encoding ScaAGO1 and ScaDCR1 respectively into the S. cerevisiae strain, W303a. A dual- reporter plasmid, encoding GFP and URA3, as well as plasmids encoding galactose-inducible silencing constructs against these re- porter genes were also integrated into W303a. The resultant strains (harboring different combinations of RNAi components) were used to measure the efficiency of silencing of either GFP using flow cytom- etry, or URA3 using growth assays (Table 1). The efficiency of GFP silencing was measured by comparing the rel- ative green fluorescence of yeast grown under non-inducing (glucose) and inducing (galactose) conditions for two different silencing con- structs [19]. Expression of the strong GFP Silencing Construct (strongSC-GFP) results in a single RNA transcript that forms a long hair- pin structure in which the ds stem region is comprised of two inverted Fig. 2. Schematic for silencing constructs directed against GFP and URA3. A. The strong silencing construct directed against GFP (strongSC-GFP) is a single transcript harboring inverted repeats of a GFP fragment and forms a dsRNA hairpin structure. The production of this construct is driven by the galactose-inducible GAL1 promoter (PGAL1) [19]. B. The weak silencing construct directed against GFP (weakSC-GFP) is comprised of two transcripts by two convergent promoters. The sense transcript (blue) is driven by the galactose-inducible GAL1 promoter (PGAL1) and the antisense transcript (red) is constitutively driven from the weaker URA3 promoter (PURA3). These two complementary tran- scripts anneal to form a dsRNA structure. C. The silencing construct directed against URA3 (SC-URA3) is a single transcript harboring inverted repeats of a URA3 fragment and forms a dsRNA hairpin structure. The production of this construct is driven by the galactose-inducible GAL1 promoter (PGAL1) and is similar to the strongSC-GFP. D. The silencing construct used in a previous study with human Dicer, Argonaute2 and TRBP was a single, antisense transcript (red) that anneals directly to the GFP mRNA to form a dsRNA structure [21]. 2676 Y. Wang et al. / Biochimica et Biophysica Acta 1833 (2013) 2673–2681
- 5. GFP segments (Fig. 2A). Expression of the weak GFP Silencing Construct (weakSC-GFP) produces a ssRNA that is perfectly complementary to another transcript driven by the URA3 promoter at the opposite end (Fig. 2B). These two promoters drive the transcription of the same cas- sette but in opposite directions which results in the formation of a long dsRNA (Fig. 2B). Consistent with previous reports, the S. cerevisiae strain express- ing both ScaAGO1 and ScaDCR1 exhibited RNAi-mediated silencing (Fig. 3A) [19]. The GFP fluorescence in yeast expressing ScaAGO1, ScaDCR1 and GFP was reduced when the expression of the weakSC- GFP construct was induced in galactose (Fig. 3A—blue line). Also con- sistent with previous studies, yeast harboring the strongSC-GFP con- struct did not show any GFP fluorescence regardless of induction conditions [19]. This suggests that the small amount of transcription from the GAL1 promoter under repressing conditions (glucose) was sufficient to completely silence GFP expression. Under non-inducing conditions, the GFP fluorescence in the strain harboring the weakSC- GFP (Fig. 3A — blue line) was identical to that of a strain that was iden- tical in every sense except that it lacked a silencing construct (Fig. 3A — filled green plot). When grown in galactose however, induction of the silencing construct resulted in a significant reduction in GFP fluores- cence (Fig. 3A). In contrast, the strain harboring the strongSC-GFP construct did not exhibit detectable GFP fluorescence even under repressing (glucose) conditions, likely due to the fact that the GAL1 pro- moter is leaky even under glucose conditions [19]. The RNAi-dependent silencing of URA3 was assayed by plating se- rial dilutions of S. cerevisiae cells on agar plates lacking uracil (-Ura), or containing uracil but also the anti-metabolite 5-fluoroorotic acid (5-FOA). The expression of the URA3 gene allows for growth on media lacking uracil but not on media containing uracil and 5-FOA, which is converted by the URA3 gene product to the toxic 5-fluorouracil. The si- lencing construct targeting URA3 (SC-URA3) contains inverted repeats of a URA3 gene fragment driven by the GAL1 promoter (Fig. 2C) [19]. The single-stranded transcript of the hpSC-URA3 construct forms a hair- pin structure that is comparable to the strongSC-GFP construct. The growth of yeast expressing ScaAGO1, ScaDCR1 and URA3 was reduced on media lacking uracil compared to control (i.e. yeast lack- ing the SC-URA3 silencing construct) only when the silencing con- struct was induced with galactose (Fig. 4). Similarly, growth of this strain on media containing 5-FOA was improved relative to control when galactose was present to induce expression of the silencing construct (Fig. 4). This shows that ScaAGO1 and ScaDCR1 are able to reduce URA3 expression by RNAi-mediated silencing. Consistent with previous reports, our assays for both GFP fluorescence and growth on media lacking uracil showed that ScaAGO1 and ScaDCR1 are able to reconstitute RNAi-mediated silencing of reporter genes in S. cerevisiae [19,20]. One major obstacle we encountered in the construction of our strains was the loss of integrated genes during successive rounds of transformation. This was likely due to the high similarity of the back- bone of the plasmids we used, which allows for unwanted homolo- gous recombination that disrupts or discards genes of interest while leaving the selectable markers intact. Moreover, no commercial anti- bodies are available for ScaAGO1 or ScaDCR1. To overcome this limi- tation, we added epitope tags to ScaAGO1 and ScaDCR1 to allow for the detection of protein expression by western blot. ScaAGO1 was tagged at the N-terminus with the hemaglutinin (HA) tag, and ScaDCR1 was tagged at the C-terminus with the myc epitope. Impor- tantly, S. cerevisiae cells expressing the epitope-tagged version of HA-ScaAGO1 and ScaDCR1-myc were capable of silencing both GFP (Fig. 3) and URA3 (Fig. 4). This demonstrated that the epitope tags do not impair the function of ScaAGO1 or ScaDCR1 and allowed us to verify the expression of these proteins in our experiments (Fig. 5). 3.2. Human Argonaute2 and S. castellii Dicer restore RNAi in S. cerevisiae While conserved in a functional sense, there are structural differ- ences in the Dicer homologues between the RNAi systems of yeast and humans (Fig. 1). To determine the level of compatibility between components like Argonaute and Dicer, we constructed yeast strains ex- pressing different cross-species, epitope-tagged pairs of these proteins. To further characterize the role of TRBP2 in RNAi, we constructed a series of yeast strains containing different combinations of human or S. castelli Argonaute and Dicer, with and without TRBP2 (Summarized in Table 1). Five strains were constructed that express the following Argonaute–Dicer pairs (with and without TRBP2): (1) HA-HsAgo2, ScaDCR1-myc; (2) HA-ScaAGO1, HsDcr1-myc; (3) HA-ScaAGO1, HsDcr1-myc, TRBP2; (4) HA-HsAgo2, HsDcr1-myc; (5) HA-HsAgo2, Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount Relative GFP Fluorescence CellCount A B C D glucose glucose glucose glucose galactose galactose galactose galactose ScaAgo1 + ScaDcr1 HA-ScaAgo1 + ScaDcr1-myc HA-HsAgo2 + ScaDcr1-myc HA-HsAgo2 + HsDcr1-myc + TRBP2 Fig. 3. GFP fluorescence in yeast expressing different pairs of Argonaute and Dicer. S. cerevisiae strains containing different Argonaute and Dicer genes from human and S. castellii. A. Yeast expressing untagged ScaAgo1 and ScaDcr1. B. Yeast expressing HA-ScaAgo1 and ScaDcr1-myc. C. Yeast expressing HA-HsAgo2 and HsDcr1-myc. D. Yeast ex- pressing HA-HsAgo2, HsDcr1-myc and TRBP2. Each panel shows four traces for GFP fluorescence in yeast grown in glucose (top) or galactose (bottom). Yeast lacking the GFP reporter (filled gray trace; A—YW3, B—YW6, C—YW9, D—YW18), yeast expressing the GFP reporter but lacking a silencing construct (filled green trace; A—YW4, B—YW7, C— YW10, D—YW19), yeast harboring both the GFP reporter and the weakSC-GFP construct (blue trace; A—YW24, B—YW26, C—YW28, D—YW30), and yeast expressing both the GFP reporter and the strongSC-GFP construct (red trace; A—YW25, B—YW27, C—YW29, D—YW31). Each strain was analyzed on a FacsCalibur flow cytometer to measure GFP fluorescence intensity. 2677Y. 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- 6. HsDcr1-myc, TRBP2. All of these strains were tested for the ability to si- lence GFP and URA3 expression. Interestingly, human Argonaute2 (HA-HsAgo2) and S. castellii Dicer (ScaDCR1-myc) restored RNAi- mediated silencing of reporter genes in S. cerevisiae (Figs. 3C and 4). Unlike the Ago–Dicer pair from S. castellii, which completely silenced GFP expression with the strongSC-GFP construct even in glucose, the HA-HsAgo2/ScaDCR1-myc pair was less efficient at silencing GFP ex- pression under these conditions (Fig. 3C). In galactose however, the HA-HsAgo2/ScaDCR1-myc pair efficiently silenced GFP expression even in strains expressing the weakSC-GFP construct. Unexpectedly, strains expressing HA-HsAgo2, HsDcr1-myc, and TRBP2, did not exhibit RNAi activity in the GFP fluorescence assay or the URA3-dependent growth assays regardless of whether the strongSC-GFP or the weakSC- GFP was employed (Figs. 3D and 4). To rule out the possibility that the epitope tags fused to human HsAgo2 and HsDcr1 interfered with func- tion, we tested strains expressing untagged versions of these proteins in the URA3 silencing assay. We verified the expression of all three com- ponents by western blot but these proteins were similarly unable to re- constitute RNAi in yeast (Supplemental Fig. 1). These data contrast with a previous study [21] which reported that expression of these human RNAi components rendered budding yeast RNAi-competent. 3.3. Human and S. castellii Argonaute proteins are subject to Hsp90 regulation Previous work in our lab and others showed that the molecular chaperone Hsp90, regulates conformational changes in Argonaute re- quired to accommodate the loading of dsRNA duplexes [23–25]. There are two Hsp90 homologues in S. cerevisiae, the constitutively expressed HSC82 and the inducible HSP82, which each share high identity with their mammalian counterparts. Importantly, human Hsp90 client proteins can be processed in vivo by the yeast Hsp90 WB: α-TRBP2 WB: α-myc WB: α-HA HA-ScaAgo1 ScaDcr1-myc HA-HsAgo2 HsDcr1-myc HsTRBP2 + + - - - - + - + - + - + - - + - - + - + + - - + - - + + + WB: α-actin 250 kDa- 130 kDa- 100 kDa- 250 kDa- 130 kDa- 100 kDa- 35 kDa- 55 kDa- Fig. 5. The detection of Argonaute, Dicer, and TRBP2 proteins by western blot. Anti-HA, anti-myc, anti-TRBP2, and anti-S. cerevisiae β-actin antibodies were used to detect pro- tein expression in yeast lysates. Strains analyzed are indicated above each lane. No RNAi components ScaAgo1 + ScaDcr1 - - + - + + HA-ScaAgo1 + ScaDcr1-myc HA-HsAgo2 + ScaDcr1-myc HA-ScaAgo1 + HsDcr1-myc HA-HsAgo2 + HsDcr1-myc HA-HsAgo2 + HsDcr1-myc+ TRBP2 - - + - + + - - + - + + - - + - + + - - + - + + - - + - + + - - + - + + Complete media Media -uracil Media +uracil +5-FOA 100 10-1 10-2 10-3 100 10-1 10-2 10-3100 10-1 10-2 10-3 W303a YW1 YW2 YW3 YW4 YW5 YW6 YW7 YW8 YW9 YW10 YW11 YW12 YW13 YW14 YW15 YW16 YW17 YW18 YW19 YW20 Fig. 4. URA3 silencing in yeast expressing different combinations of Argonaute and Dicer measured with a growth assay. S. cerevisiae strains containing different Argonaute and Dicer genes from human and S. castellii. Each group has three strains; yeast lacking both the pRS406-PADH1GFP(S65T) reporter construct and the pRS403-PGAL1-SC-URA3 silencing con- struct, yeast harboring only the reporter construct, and yeast harboring both the reporter and silencing constructs. Each strain was grown on media containing uracil (left), media lacking uracil (centre), and media containing uracil and 5-FOA (right). Cells were grown overnight in appropriate media and then diluted to 1 × 108 cells per ml. 10-fold serial dilutions were prepared and 5 μl aliquots were spotted on indicated plates and grown for 2 days at 30 °C. 2678 Y. Wang et al. / Biochimica et Biophysica Acta 1833 (2013) 2673–2681
- 7. system. We therefore questioned whether the functions of human and S. castellii Argonautes in the reconstituted RNAi pathways were regulated by Hsp90. To determine this, we employed the URA3-dependent growth assay in the presence of the potent Hsp90 inhibitor, radicicol [32]. Under conditions where silencing constructs were expressed, strains expressing S. castellii Dicer and either human or S. castellii Argonaute showed reduced silencing of URA3 (i.e. increased growth relative to control strain lacking a silencing construct) when grown on plates lacking uracil but supplemented with radicicol (Fig. 6). This suggests that Hsp90 regulates both human and S. castellii Argonaute function in RNAi. 4. Discussion Since the discovery of RNAi, a great deal of research has focused on the mechanism and biological significance of this conserved pathway. Clues regarding how individual steps are regulated have emerged but much remains unknown. In the present study, we provide evidence that Argonaute proteins in the most rudimentary RNAi systems are regulated by the Hsp90 chaperone system. This is consistent with the growing body of evidence suggesting that a significant conforma- tional change must occur in Argonaute proteins to accommodate dsRNAs [33–35]. Hsp90 is known to regulate similar conformational changes in steroid hormone receptors that allow for ligand binding [36]. While it is possible Hsp90 regulates the S. castellii Dicer that is common in both reconstituted systems, this seems unlikely in light of the studies on the Drosophila RNAi system [23,24]. Specifically, these reports show that blocking Hsp90 activity does not affect Dicer binding to, or processing of, dsRNA precursors but severely im- pairs Argonaute binding to Dicer products. We were surprised that human Dicer, even with its cofactor TRBP2, was not able to reconstitute RNAi in S. cerevisiae with either S. castellii or human Argonaute given a recent report to the contrary [21]. The explanation for this discrepancy may lie with the nature of the silencing constructs we used in our study [19]. Both the strong and weak silencing constructs we employed are double-stranded; the weak silencing construct is comprised of two complementary transcripts and the strong silencing construct is a single transcript with internal complementarity that results in a double-stranded hair- pin structure (Fig. 2A–C) [19]. In contrast, the silencing construct used with human Dicer, Argonaute2 and TRBP2 by Suk and colleagues is a single-stranded RNA molecule that must anneal directly to the GFP mRNA to form a double-stranded structure (Fig. 2D) [21]. This difference could have a profound impact on how RNAi components are recruited to the dsRNA structure and on how it is processed. Regardless, it seems that in our assays, human Dicer, TRBP2 and Argonaute2 are not able to process bona fide RNAi precursors to gen- erate functional RISC. Several studies illustrate the importance of the 5′ end and the 3′ overhang structures that result from cleavage of pre-miRNAs by the microprocessor complex in the nucleus for subsequent processing by canonical Dicer proteins [7,8,10]. Canonical Dicer proteins bind to the 3′ overhang generated by microprocessor cleavage via the PAZ domain. Dicer then acts as a “molecular ruler” and cleaves at a fixed distance from this PAZ domain via its two RNAseIII domains and generates the characteristic 21–23 nucleotide dsRNA product. Importantly, the silencing constructs we used have large portions of No RNAi components ScaAgo1 + ScaDcr1 - - + - + + HA-ScaAgo1 + ScaDcr1-myc HA-HsAgo2 + ScaDcr1-myc HA-ScaAgo1 + HsDcr1-myc HA-HsAgo2 + HsDcr1-myc HA-HsAgo2 + HsDcr1-myc+ TRBP2 - - + - + + - - + - + + - - + - + + - - + - + + - - + - + + - - + - + + Media + DMSO Media -uracil + DMSO Media + 75µM RD Media -uracil + 75µM RD 100 10-1 10-2 10-3 100 10-1 10-2 10-3 100 10-1 10-2 10-3 100 10-1 10-2 10-3 W303a YW1 YW2 YW3 YW4 YW5 YW6 YW7 YW8 YW9 YW10 YW11 YW12 YW13 YW14 YW15 YW16 YW17 YW18 YW19 YW20 Fig. 6. Hsp90 inhibitor radicicol alleviates URA3 silencing. S. cerevisiae strains containing different Argonaute and Dicer genes from human and S. castellii. Each group has three strains; yeast lacking both the pRS406-PADH1GFP(S65T) reporter construct and the pRS403-PGAL1-SC-URA3 silencing construct, yeast harboring only the reporter construct, and yeast harboring both the reporter and silencing constructs. Each strain was grown on media containing uracil and DMSO control (left), media containing uracil and 75 μM radicicol (centre left), media lacking uracil with DMSO control (centre right), and media lacking uracil with 75 μM radicicol (right). Cells were grown overnight in appropriate media and then diluted to 1 × 108 cells per ml. 10-fold serial dilutions were prepared and 5μl aliquots were spotted on indicated plates and grown for 2 days at 30 °C. 2679Y. Wang et al. / Biochimica et Biophysica Acta 1833 (2013) 2673–2681
- 8. non-complementary single-stranded RNA beyond the regions of complementarity that may not be recognized by the PAZ domain of human Dicer or, if they are, are too far from the double-stranded portion of the molecule to result in cleavage. In contrast, non-canonical Dicer proteins from yeast lack a PAZ domain (Fig. 1) and are thought to bind to dsRNA substrates as homodimers [37]. Polymerization of these Dicer homodimers along the length of the dsRNA is thought to facilitate coordinated cleavage required to generate products 21–23 nt in length independent of the structure of the substrate ends. This “inside–out” cleavage mechanism may allow for the functional versatility associated with non-canonical Dicer proteins as they are known to play an impor- tant role in ribosomal and spliceosomal RNA processing [38]. In contrast to Dicer proteins from yeast and humans, Argonaute proteins are structurally much more conserved. Argonaute proteins from humans, S. castellii, and the fission yeast S. pombe share all four major domains (Fig. 1A). Moreover, two recent publications [20,35] showed the crystal structures of human and K. polysporus Argonaute are very similar. Both proteins adopt a bilobed shape with an internal cleft to bind dsRNA duplexes. Interestingly, the different domains of Argonaute2 all appear to bind to miRNA in a fashion that is conserved with other Argonaute homologues [33]. Moreover, Argonaute2 is rendered protease resistant upon miRNA binding suggesting that the ligand-free protein is less stable and per- haps more conformationally dynamic that the bound form [33]. This is important because rapid conformational sampling is one of the few hallmarks of Hsp90 client proteins. The oncoprotein Bcr–Abl is a well characterized Hsp90 client protein for which drugs are avail- able that bind to and stabilize both the active and inactive states [39]. Intriguingly, stabilization of Bcr–Abl in either conformation weakens its interaction with Hsp90. Therefore structurally dynamic, but not necessarily misfolded, proteins are likely to be Hsp90 clients. Hormone receptors are useful comparisons to Argonaute as an Hsp90 client as well. In the absence of ligand, estrogen and glucocorticoid re- ceptors form complexes with Hsp90 in the cytoplasm in a ligand-free state. Hormone binding requires Hsp90 and results in a ligand-bound but Hsp90-free complex that translocates to the nucleus to activate downstream transcription [40]. Similar to what is observed with hor- mone receptors, ligand binding (miRNA in the case or Argonaute) re- quires Hsp90 and also results in stabilization of the protein. 5. Conclusions In summary, this study presents a powerful model for studying in- dividual RNAi components in a minimal system. 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