Exon 3
1201 CGAGCGGCTCTTTACGATCGCCTGCTCGATACTCAACTACCGCGAGATGGGCAAGGGCAAAAGCAAGAAGATAGCCAAGCGCTTGGCCGCCCACCGCATG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
97 CGAGCGGCTCTTTACGATCGCCTGCTCGATACTCAACTACCGCGAGATGGGCAAGGGCAAAAGCAAGAAGATAGCCAAGCGCTTGGCCGCCCACCGCATG
E
R
L
F
T
I
A
C
S
I
L
N
Y
R
E
M
G
K
G
K
S
K
K
I
A
K
R
L
A
A
H
R
M
1301 TGGATGCGTCTGCAGGAGACTCCCATCGATTCGGGCAAAATCAGCGACAGCATCTGCGGCGAGTTGGAGGGCGAAGTGAGTATCATTCAAGACATCGATC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
197 TGGATGCGTCTGCAGGAGACTCCCATCGATTCGGGCAAAATCAGCGACAGCATCTGCGGCGAGTTGGAGGGCGAAGTGAGTATCATTCAAGACATCGATC
W M R L Q E T P I D S G K I S D S I C G E L E G E V S I I Q D I D R
1401 GTTACGAGCAAGTCTCTAAAGATTTTGAGTTCATCAAGATCTAACCCACAAGCAATTTTCTAATATTTTTAGTGCAATTATTTTGAATGCAGTCCCATGC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
297 GTTACGAGCAAGTCTCTAAAGATTTTGAGTTCATCAAGATCTAACCCACAAGCAATTTTCTAATATTTTTAGTGCAATTATTTTGAATGCAGTCCCATGC
Y E Q V S K D F E F I K I *
1501 TTTAGTTTATAAAATTTTAAAAATTGATTTTATCAAATATATGTCATAAATGTTATATATTATTTAATACGTTTGTCCTCTATCCCTTCTCAAATGTAAT
|||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
397 TTTAGTTTATAAAATTTTAAAAATTGTTTTTATCAAATATATGTCATAAATGTTATATATTATTTAATACGTTTGTCCTCTATCCCTTCTCAAATGTAAT
1601 GTAGAACGTTTAGAGTAATCCTCAGTTGTAAATATCCCTATAATCTTTTAAATTATGTATCTAGCTAGAGTTTTAGTACTATAGCATCCCTTTTATAGTG
||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||
497 GTAGAACGTTTAGAGTAATCCTCAGTTGTAAATATCCCTATAATCTTTTTAATTATGTATCTAGCTAGAGTTTTAGTACTATAGCATCCCTTTTATAGTG
1701 TGCTATCTGATATTTTTTATTGTCCTTGTTTTTATAACTAATGTTTTTGACATAATTATACATATAATTAACATAATGCTTACACCTTATTTATATTTCT
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
597 TGCTATCTGATATTTTTTATTGTCCTTGTTTTTATAACTAATGTTTTTGACATAATTATACATATAATTAACATAATGCTTACACCTTAAAAAAAAAAAA
Supplementary Figure 1: loqs-RD specific sequence
Top strand: Drosophila melanogaster genome sequence;
Bottom strand: Cloned 3’-RACE sequence; the appended poly-A tail is colored in green
bd.
inp.
GFP
bd.
inp.
bd.
bd.
inp.
Loqs PB Loqs PD R2D2
inp.
myc-tagged
construct:
Dcr-1 Dcr-2 -
PB PD R2D2GFP
GFP
bd.
bd.
R2D2
inp.
bd.
Loqs PD
inp.
Loqs PA
bd.
bd.
inp.
Loqs PB
inp.
myc-tagged
construct:
Dcr-1 Dcr-2 -
PB PA -PD
R2D2GFP
GFP
bd.
bd.
R2D2
inp.
bd.
Loqs PD
inp.
bd.
Loqs PA
inp.
bd.
Loqs PB
inp.
myc-tagged
construct:
Dcr-1 -
Dcr-2 PB PA -PD
R2D2GFP
Supplementary Figure 2: Co-immunoprecipitation of Dcr-2 with tagged Loqs-PD
Three independent experiments are shown. Although the extent of association varied,
Loqs-PD associates preferentially with Dcr-2. While some association between Loqs
PA/PB and Dcr-2 may also occur, these two isoforms preferentially associate with Dcr-1.
2
fold change tub-Gal4 / empty Gal4
250
abundance rel. to RP49 mRNA
50
229,45
200
149,58
150
100
55,16
50
1,66
1,89
0
empty Gal4
1,45
tub.-Gal4
empty Gal4
tub.-Gal4
45,93
40
30
20
10
3,575
0
0,065
0,2
0,01
1,53
0,388
0,658
0,311
0,644
1,243
0,45
Suppl. Figure 3:
Induction of transcription measured with the RNA-fractionation protocol
Cells were transfected with a UAS-GFP plasmid and either a promotorless Gal4-plasmid
(pPTGal4 empty) or a tubulin-promotor Gal4-Plasmid (pPTGal4 with tubulin-promotor).
Two days post transfection, the RNA was pulse-labeled, extracted, fractionated and
quantified by qRT-PCR as described in the manuscript.
3
Top panel:
Changes in the mRNA levels of UAS-GFP and endogenous GAPDH between a
promotorless Gal4-Plasmid and the tubulin-promotor Gal4-Plasmid; an increased mRNAabundance can be seen in all RNA fractions. Theoretically, the increase in total mRNA
should lie between the values for the < 1h and the > 1h fraction, as is the case for
GAPDH. The apparent discrepancy for GFP appears to be due to a concomitant increase
in mRNA stability (see below), resulting in an exaggerated ratio for the > 1h fraction.
Bottom panel:
For each individual measurement, the mRNA abundance in the total RNA fraction lies
between the abundance in the newly transcribed (< 1h) and the old (> 1h) RNA.
However, the contribution of old RNA to the total RNA pool is particularly low for GFP
in the uninduced state (see table below). This ratio represents the mRNA half-life relative
to rp49 (<1: shorter than rp49, >1: longer than rp49), and this result implies that
degradation of the GFP mRNA is slower when it is very abundant. One possible
explanation could be the saturation of a specific degradation pathway upon the strong
induction via the tubulin-promotor Gal4-plasmid.
GFP unind.
GFP ind.
GAPDH unind.
GAPDH ind.
old/total
0,15
0,43
0,79
0,70
4
63N1 library
naïve cell library
16000
pKF63
corresponding reads
corresponding reads
16000
12000
8000
pKF63
12000
8000
4000
4000
0
0
20 nt
21 nt
22 nt
20 nt
23 nt
600
400
200
22 nt
23 nt
21 nt
22 nt
23 nt
CG4068
CG4068
corresponding reads
corresponding reads
600
21 nt
400
200
0
0
20 nt
22 nt
20 nt
23 nt
250000
Dme transposons
corresponding reads
corresponding reads
250000
21 nt
200000
150000
100000
50000
Dme transposons
200000
150000
100000
50000
0
0
20 nt
21 nt
22 nt
20 nt
23 nt
21 nt
22 nt
23 nt
Supplementary Figure 4: Length distribution of small RNA reads
The length distribution of small RNAs matching the indicated targets was calculated
based on the BOWTIE output file. In the case of CG4068, reads matching to the entire
hairpin sequence (i.e. not only the most prominent sequence) were considered.
5
reads / 10 bp window
500
S2-cells, mock treated
2 50
0
-25 0
-50 0
dsRNA
GFP
pos. (nt):
reads / 10 bp window
50 0
0
2000
white
4000
6000
pUC-ori AmpR
8000
10000
S2-cells, -eliminated
25 0
0
-2 5 0
-5 0 0
Supplementary Figure 5: Enrichment of transgene-derived endo-siRNAs upon elimination
Ago2-loaded small RNAs are modified and thus resistant to -elimination at their 3’-end.
Only the resistant RNAs will be cloned and sequenced after such a treatment, therefore
enrichment in a-eliminated library is an indication that the small RNAs were loaded
into Ago2. The top panel shows the same data as in Fig. 4B of the manuscript but the
scale was changed in order to visualize the enrichment in the-eliminated library.
6