Supporting Information
Duan et al.
Table of contents:
1. Supplementary Figures Legends S1-13
2. Supplementary Methods S1-3
3. Supplementary Tables S1-4
4. Supplementary References
1. Supplementary Figure Legends
S1. Diagram of detection of the 1° and 2° siRNAs with the probes designed based on the
pebble (1) gene sequence using Northern hybridization and for the RNAse Protection
Assay. The red fragment represents the sequence targeted by hair-pin RNA; the blue
fragment represents the sequence upstream of the targeted sequence. Probe 1 and 2 as
indicated were for Northern blotting and Hybridization, while probe 3 and 4 were for RNAse
protection assay. Probe 1 and 4 were for 1° siRNAs detection; probe 2 and 3 were for 2°
siRNAs detection.
S2. pebble gene cDNA sequence. The sequence targeted by the dsRNA hairpin pblRNAi.4B
is shown in red (2)
S3. Detection of 1° and 2° siRNAs from EGFP translational fusions. This diagram shows
the probes designed based on the egfp gene sequence (Clontech) (3) and upstream-linked syt (
Genebank accession No. M55048) (4) or NaChBac sequence (kindly provided by Dr. Dejian
Ren) (5) through Northern hybridization.
1
S4. NaChBac Insert Sequence. EcoRI (GAATTC) & XbaI (TCTAGA) sites are underlined.
Translation start (ATG) and stop (TGA) codons are in bold.
S5. Evolutionary relationships of metazoan animals and the distribution of RdRp genes.
The metazoan phylogeny is compiled from (6),(7) and (8). The available genomes were
screened for the presence of the RdRp gene (9, 10). Organisms whose names are underlined
retain the gene; those not underlined have lost it. See (10) for details of the sequences
identified as RdRp genes.
S6. rrf-1 enhances eGFP silencing in a dsRNA –dependent manner while EGO-1conferred silencing is dsRNA-independent (refer to Fig 2).The crosses are the same as Fig
2 except that in this Figure, the UAST-syt.eGFP is located on chromosome II while
eGFP.dsRNA.R is on chromosome III.
S7. Cross to test direct silencing by superfly w; da-Gal4; UAST-ego-1 is reversible or
irreversible.
S8. Superfly w; da-Gal4; UAST-ego-1 silenced general driver da-Gal4 activated hairpin
pblRNAi 4B(3.1), therefore rescued the progeny which otherwise would be killed at the
pupal stage.
S9. ego-1 can independently silence hairpin gene only under the control of UAST, which
is strong for somatic cell expression, rather than UASP, which is for germline and
somatic cell expression. Three parallel crosses were set up as follow to test if RRF-1 and
EGO-1 can independently silence hairpin pblRNAi 4B(3.1).‫٭‬Please note: Cross in “C” was
doubled in order to examine more progeny
2
S10. EGFP mRNAs detections with Northern blotting and Hybridization.~10 µg of each
total RNA sample was running on the 1.2% Formaldehyde gel under ~80V for ~2h, followed
by overnight blotting onto Amersham HybondTM-N+ membrane (GE Healthcare), and the
membrane was hybridized with EGFP DNA probe labelled with Ready-to-go DNA labelling
Beads (-dCTP) (Amersham Biosciences), finally exposed and developed
using a
PhosphorImager as before.
S11A-B. qRT-PCRs quantify rrf-1 and ego-1 expression. To demonstrate that RdRp itself
was expressed; we performed qRT-PCR to quantify the mRNAs of the transgenes in various
transgenic flies. Each reaction had 4 replicates. A normalized value for relative ego-1
expression in each line was obtained by calculating the mean from the 4 replicates minus the
mean values of no RT controls, then dividing by the actin mean value.
S12 A & B: Schematic diagrams of restriction sites of HpaII and HhaI in EGFP coding
region (A), and transgenes’ promoter region (B), as well as its adjacent sequences,
respectively. Red fragments denote the fragment in EGFP or promoter detected by the
probes; the green fragments denote the adjacent plasmid sequence (upstream) or the fly
genome sequence (downstream) (A)/ transgenes’ (syt-eGFP or ego-1) sequences
(downstream) (B).
S13. Promoter region sequence alignment. Seq29 (S29) sequence was detected methylated
in the region comprised of 5 x Gal4 UAS and the first half of the HS promoter.
Supplementary Methods
S1. Crosses that set up for silencing of the Drosophila endogenous gene pebble with or
without RdRps in Fig 1.
A: w; En-Gal4; + x w; pblRNAi4B; +
B: w; En-Gal4; P-ego-1 x w; pblRNAi4B; +
C: w; En-Gal4/CyO; T-rrf-1 x w; pblRNAi4B; +
3
↓
Select progenies without CyO
D: w; En-Gal4; T-ego-1 x w; pblRNAi4B; +
↓
Wild type phenotype (picture not shown)
S2. Crosses to obtain fluorescence or fluorescence-silenced larvae in Fig. 2
A: w; da-Gal4; + x w; +; UAS-syt.EGFP
B: w; da-Gal4; + x w; UAS-GFP.dsRNA.R; UAS-syt.eGFP
C: w; da-Gal4; T-rrf-1 x w; UAS-GFP.dsRNA.R; UAS-syt.eGFP
D: w; da-Gal4; T-rrf-1 x w; +; UAS-syt.eGFP
E: w; da-Gal4; T-ego-1 x w; UAS-GFP.dsRNA.R; UAS-syt.eGFP
F: w1118
G: w; da-Gal4; T-ego-1 x w; +; UAS-syt.eGFP
H: w; da-Gal4; + x w; UAS-NaChBac-EGFP; +
I: w; da-Gal4; + x w; UAS-NaChBac-EGFP ; UAS-GFP.dsRNA.R
J: w; da-Gal4; T-rrf-1 x w; UAS-NaChBac-EGFP ; UAS-GFP.dsRNA.R
K: w; da-Gal4; T-rrf-1 x w; UAS-NaChBac-EGFP ; +
L: w; da-Gal4; T-ego-1 x w; UAS-NaChBac-EGFP ; UAS-GFP.dsRNA.R
M: w; da-Gal4; T-ego-1 x w; UAS-NaChBac-EGFP ; +
N: w; da-Gal4; + x W‫ ;٭‬UAS-Rab4-mRFP; +
O: w; da-Gal4; T-rrf-1 x W‫ ;٭‬UAS-Rab4-mRFP; +
P: w; da-Gal4; T-ego-1 x W‫ ;٭‬UAS-Rab4-mRFP; +
4
S3. Crosses to obtain fluorescence or fluorescence-silenced adult flies in Fig. 5
A: w; da-Gal4; UAS-syt.EGFP/Tm6b x w; T-ego-1; UAS-syt.EGFP
↓
w; da-Gal4/T-ego-1; UAS-syt.EGFP/Tm6b
B: w; da-Gal4; UAS-syt.EGFP/Tm6b x w; T-ego-1; UAS-syt.EGFP
↓
w; da-Gal4/T-ego-1; UAS-syt.EGFP
Supplementary Tables
Table S1. Fly strains from Ozdros (Australia)
Genotype
Function
Chromosom
e
Homozygo
us
W; UAS-pblRNAi.4B;+
dsRNA targeting pebble gene
II
Yes
dsRNA targeting egfp gene
II
Yes
W; +; UAS-GFP.dsRNA.R
dsRNA targeting egfp gene
III
Yes
W; Da-Gal4;+
General driver
Tissue specific driver for
activation on the posterior
compartment of the wing disc
II
Yes
II
Yes
W; UAS-GFP.dsRNA.R; +
W; En-Gal4;+
5
Table S2. Fly strains from Bloomington (USA)
Genotype
Function
Bloomington ID
Number
Chromosome
Homozygous
W‫ ;٭‬+; UASsyt.eGFP
Synaptotagmin gene
(syt) fused to egfp gene
BL6926
III
Yes
Y1W‫ ;٭‬UASNaChBac-EGFP;
+
Bacteria NaChBac
gene fused to egfp gene
BL9466
II
Yes
W‫ ;٭‬UAS-Rab4mRFP; +
Rab4 fused to rfp gene
BL8505
II
Yes
Table S3. Transgenic flies created in this study for egfp gene silencing in Fig 2 and Fig 3.
Genotype
Function
Bloomington stock
used
Homozygous
W; da-Gal4; UAST-ego-1
Da-Gal4 General
activation of ego-1
No
Yes
W; da-Gal4; UAST-rrf-1
Da-Gal4 General
activation of rrf-1
No
Yes
W; UAS-GFP.dsRNA.R;
UAS-syt.eGFP
egfp dsRNA for
targeting syt.eGFP
W; UASNaChBac.EGFP; UASGFP.dsRNA.R
egfp dsRNA for
targeting
NaChBac.EGFP
Tissue specific driver
for activation rrf-1
on the posterior
compartment of the
wing disc
Tissue specific driver
for activation ego1on the posterior
compartment of the
wing disc
Tissue specific driver
for activation ego1on the posterior
compartment of the
wing disc
W; En-Gal4/CyO; T-rrf-1
W; En-Gal4; T-ego-1
W; En-Gal4; P-ego-1
6
Yes/GFP.dsRNA.R
combined with
BL6926
Yes/GFP.dsRNA.R
combined with
BL9466
Yes
Yes
No
No
No
Yes
No
Yes
Table S4. PCR primers for Probes used for Northern Hybridization or RNAse
protection assay
For Northern
Hybridization
Probe ID
Primer Sequences for probe preparation
Pebble
Probe 1
For:CGATATCGAAGGATGTCCG
Rev:GCATGCTTGCACAGTCCT
√
Pebble
Probe 2
For:TCGATTGTACCACCAGTCC
Rev:GAATATATCGAAGATGGCCA
√
Pebble
Probe 3
Pebble
Probe 4
syt sense
probe
syt antisense
probe
NaChBac
sense
probe
NaChBac
anti-sense
probe
For:TAATACGACTCACTATAGGGAGGTGCCC
AGTATCAGTCTGC
√
Rev:GAATATATCGAAGATGGCCA
For:TAATACGACTCACTATAGGGAGGCTTTA
ACAAAACGCCCAAC
Rev:GCATGCTTGCACAGTCCT
For:TAATACGACTCACTATAGGGAGGACTGT
CTGATCCATATGTG
Rev:AATACGATCGTAGTCCACC
For:TAATACGACTCACTATAGGGAGGAATAC
GATCGTAGTCCACC
Rev: ACTGTCTGATCCATATGTG
For:TAATACGACTCACTATAGGGAGGCGGC
GTTAGGAAACATCTT
Rev:CTTCGGCAAAAATTGGTCGC
For:TAATACGACTCACTATAGGGAGGCTTCG
GCAAAAATTGGTCGC
Rev: CGGCGTTAGGAAACATCTT
For:TAATACGACTCACTATAGGGAGGAGTTC
egfp sense GAGGGCGACACC
probe
Rev:TCGATGTTGTGGCGGATC
For:TAATACGACTCACTATAGGGAGGTCGAT
egfp antiGTTGTGGCGGATC
sense
probe
Rev: AGTTCGAGGGCGACACC
7
For RNAse
protection
assay
√
√
√
√
√
√
√
Note: The red sequences in the table are the T7 RNA polymerase promoter adapted from
HybspeedTM RPA kit (Ambion).
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4.
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5.
Ren D, et al. (2001) A prokaryotic voltage-gated sodium channel. Science 294:23722375.
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