Supplemental Figures

(1)

Nephronophthisis gene products display RNA-binding

proper7es and are recruited to stress granules

Luisa Estrada Mallarino

1,2

_{, Chris4na Engel}

1

_{, İbrahim Avşar Ilık}

3†

_{, Daniel Ma4czka}

4

_{, Florian Heyl}

4

_,

Barbara Müller

1

_{, Toma A. Yakulov}

1

_{, Jörn Dengjel}

5+

_{, Rolf Backofen}

4

_{, Asifa Akhtar}

3,6

_{, Gerd}

Walz

1,7

_*

1

_{Renal Division, University Freiburg Medical Center, Faculty of Medicine, University of Freiburg,}

Germany

2

_{Faculty of Biology, University of Freiburg}

3

_{Max Planck Ins4tute of Immunobiology and Epigene4cs, Freiburg, Germany}

4

_{Ins4tute for Informa4cs, Albert-Ludwigs-University, Freiburg, Germany}

5

_{Department of Dermatology, Medical Center, and Freiburg Ins4tute for Advanced Studies,}

University of Freiburg, Germany

6

_{CIBSS - Centre for Integra4ve Biological Signalling Studies, University of Freiburg}

7

_{Signalling Research Centres BIOSS and CIBSS, University of Freiburg}

(2)

Supplemental Figure 1

BICC1

INV

ANKS3

(3)

Supplemental Figure 2

GO-Term Term Description Observed Gene _Count Background Gene _Count FDR Biological Process

GO:0043604 amide biosynthetic process 66 414 1.01e-60 GO:0006412 translation 61 313 1.43e-60 GO:0006518 peptide metabolic process 63 440 1.45e-55 GO:0043603 cellular amide metabolic process 68 644 1.46e-52 GO:1901566 organonitrogen compound biosynthetic process 73 1122 2.74e-43

Molecular Function

GO:0003735 structural constituent of ribosome 58 153 3.78e-72 GO:0005198 structural molecule activity 70 546 5.04e-60 GO:0003723 RNA binding 66 986 1.40e-39 GO:1901363 heterocyclic compound binding 106 4748 3.66e-26 GO:0097159 organic cyclic compound binding 106 4818 1.03e-25

Cellular Component

GO:0022626 cytosolic ribosome 58 107 1.69e-79 GO:0044391 ribosomal subunit 60 181 2.82e-72 GO:0005840 ribosome 61 215 3.04e-70 GO:0044445 cytosolic part 59 228 7.80e-66 GO:1990904 ribonucleoprotein complex 78 765 2.34e-61

KEGG

mmu03010 Ribosome 55 128 3.76e-71 mmu01200 Carbon metabolism 8 118 0.00052 mmu05322 Systemic lupus erythematosus 7 92 0.00064 mmu00020 Citrate cycle (TCA cycle) 4 32 0.0063 mmu00010 Glycolysis / Gluconeogenesis 5 65 0.0065

a

b

100 kD

75 kD

150 kD

100 kD

75 kD

150 kD

-ant

i-AN

KS6

Co

ntr

ol

- ANKS6

Immunoprecipitation

Lysates

(4)

Supplemental Figure 3

a

b

75 kD - 100 kD - 150 kD - 50 kD 37 kD

-Co

nt

ro

l

BI

C

1 IN

V

AN

KS3

AN

KS6

BBS3

AR

L1

3B

Lysates: anti-Flag 75 kD -150 kD - 100 kD - 50 kD 37 kD

-Affinity Purification: cobalt/streptavidine WB: anti-Flag

(5)

Supplemental Figure 4

BICC1

INVERSIN

(6)

Supplemental Figure 5

BICC1

INVERSIN

(7)

Supplemental Figure 6

BICC1

ANKS6

INVS ANKS3

5 RP11 4 POLR2A 6 NUCKS1 7 SLC38A2 8 SMC1A 24 ATP2A2 23 AFF4 24 CALR 26 CCNT1 27 EIF4G1 28 EIF4G2 29 ERC1 30 HNRNPA0 31 MDM2 32 MIDN 34 PRKAR1A 35 SRSF2 36 TFRC 37 TMTC3 20 CSNK1E 18 CANX 22 PP1CC 16 LBR 14 C5orf51 10 PMAIP1 9 MARCH6 12 RC3H1 13 SYNCHRIÜ 3 POLR2A 52 ND4 45 HIST1H1E 66 SCARNA2 51 MIF 39 ATP5F1B 63 RPS16 41 ENO1 46 HNRNPA2B1 42 GNB2L1 53 NEAT1 50 MALAT1 40 CYTB 67 SNHG1 64 RPPH1 62 RPL7A 69 SNRPB 44 GSK3A 43 GPI 47 HNRNPUL1 48 HSPA8 54 NP56 55 PTMS 56 RCC1 57 RNR1 58 RPL13A 59 RPL23A 60 RPL3 61 RPL4 65 RPS8 68 SNH65 70 TMBIM6 38 ARHGDIA 49 KHSRP 33 ND5 17 ZNF703 21 MAZ 15 GAS5 11 RACK1 19 CKB 2 EEF2 1 XIST Column Z-Score

(8)

Supplemental Figure 7

BICC1

INVERSIN

ANKS3

ANKS6

mRNA metabolism primary metabolism cytoplasmic translation

organic substance catabolism

cytoplasmic translation

organic substance catabolism

negative regulation of biological process

mRNA metabolism

a

b

_ANKS6

150 bp

-a-Flag no Ab

10%

input

RT

+ -

+ +

-a-Flag no Ab

10%

input

+ -

+ +

-a-Flag no Ab

10%

input

ANKS3

INVERSIN

+ -

+ +

(9)

-Supplemental Figure 8

100 100 75 150 100 75 75 150 150 50 37 25

kD-F.

A

N

K

S

3

F. A

N

K

S

6

F. IN

V

E

R

S

IN

F. B

IC

C

1

F. B

B

S

3 V5.AGO2

- V5.AGO2

- F.INVERSIN

- F.BICC1

- F.ANKS6

- F.ANKS3

- F.BBS3

IP: Anti-Flag, WB: anti-V5

Lysates, WB: anti-V5

IP: Anti-Flag, WB: anti-Flag

100 100 150 100 75 150 150 50 37 25

kD-F.

A

N

K

S

3

F. A

N

K

S

6

F. IN

V

E

R

S

IN

F. B

IC

C

1

F. B

B

S

3 CAPRIN1.GFP

- CAPRIN1.GFP

- F.INVERSIN

- F.BICC1

- F.ANKS6

- F.ANKS3

- F.BBS3

IP: Anti-Flag, WB: anti-V5

Lysates, WB: anti-V5

IP: Anti-Flag, WB: anti-Flag

- CAPRIN1.GFP

(10)

Supplemental Figure 9

control 0.4 pmol

500 μm

ago2 SBM 0.4 pmol

a

48 hpf

b

48 hpf

control 1.2 pmol

500 μm

ago2 SBM 1.2 pmol

c

48 hpf

d

48 hpf

n=138

n=164

n=0

n=187

0 5 10 15 20 25 30 35 40

control

MO 0.4

pmol

ago2

SBM 0.4

pmol

control

MO 1.2

pmol

ago2

SBM 1.2

pmol

% cyst fo

rmatio

n

n.s.

p = 0.004

n=214

n=192

n=233

0 5 10 15 20 25 30 35 40 45 50

anks3 SBM

0.4 pmol

Control MO

0.8 pmol

ago2 SBM

0.8 pmol

Control MO

0.4 pmol

anks3 SBM

0.4 pmol

ago2 SBM

0.8 pmol

% cyst fo

rmation

p = 0.005

p = 0.001

100 μm

48 hpf

ago2 SBM

anks3 SBM

control MO

e

f

g

h

(11)

BICC1

TIA-1

merge

HIPP

DMSO

AGO2

TIA-1

merge

DMSO

HIPP

a

b

Supplemental Figure 10

2D intensity histogram 256.0 x 256.0 % zero-zero pixels: 1.13 % saturated ch1 pixels: 4.84 % saturated ch2 pixels: 0.01

Warning! y-intercept high - The absolute y-intercept of the auto threshold regression line is high. Maybe you should use a ROI, maybe do a background subtraction in both channels.

m (slope): 0.52 b (y-intercept): 8.31 b to y-max ratio: 0.05 Ch1 Max Threshold: 7.00 Ch2 Max Threshold: 12.00

Pearson's R value (no threshold): 0.38 Pearson's R value (below threshold): -0.00 Pearson's R value (above threshold): 0.21 Li's ICQ value: 0.204

Spearman's rank correlation value: 0.51863702 Spearman's correlation t-statistic: 631.7182 t-statistic degrees of freedom: 1084542.000 Manders M1 (no threshold): 1.000

Manders M2 (no threshold): 0.974 Manders M1 (threshold): 0.825 Manders M2 (threshold): 0.722

Kendall's Tau-b rank correlation value: 0.3824

Channel 1 Ch a n n e l 2 Pearson‘s R = 0.21 2D intensity histogram 256.0 x 256.0

Warning! zero-zero ratio too high - The ratio between zero-zero pixels and other pixels is larger 0.14. Maybe you should use a ROI.

Warning! saturated ch1 ratio too high - The ratio between saturated pixels and other pixels in channel one is larger 0.10. Maybe you should use a ROI.

Warning! saturated ch2 ratio too high - The ratio between saturated pixels and other pixels in channel two is larger 0.10. Maybe you should use a ROI.

% zero-zero pixels: 14.02 % saturated ch1 pixels: 13.85 % saturated ch2 pixels: 6.40 m (slope): 1.62 b (y-intercept): 1.27 b to y-max ratio: 0.01 Ch1 Max Threshold: 1.00 Ch2 Max Threshold: 3.00

Spearman's rank correlation value: 0.68489898 Spearman's correlation t-statistic: 715.3560 t-statistic degrees of freedom: 579182.000 Manders M1 (no threshold): 0.999 Manders M2 (no threshold): 0.900 Manders M1 (threshold): 0.994 Manders M2 (threshold): 0.892 Channel 1 Ch a n n e l 2 Pearson‘s R = 0.91 2D intensity histogram 256.0 x 256.0 % zero-zero pixels: 0.21 % saturated ch1 pixels: 4.46 % saturated ch2 pixels: 2.22

m (slope): 1.72 b (y-intercept): -6.48 b to y-max ratio: -0.04 Ch1 Max Threshold: 5.00 Ch2 Max Threshold: 2.00

Channel 1 Ch a n n e l 2 Pearson‘s R = 0.90

Statistics

2D intensity histogram 256.0 x 256.0

Warning! saturated ch1 ratio too high - The ratio between saturated pixels and other pixels in channel one is larger 0.10. Maybe you should use a ROI.

Warning! saturated ch2 ratio too high - The ratio between saturated pixels and other pixels in channel two is larger 0.10. Maybe you should use a ROI.

% zero-zero pixels: 1.70 % saturated ch1 pixels: 10.33 % saturated ch2 pixels: 0.28

m (slope): 1.08 b (y-intercept): -8.81 b to y-max ratio: -0.05 Ch1 Max Threshold: 15.00 Ch2 Max Threshold: 7.00

Pearson's R value (no threshold): 0.59 Pearson's R value (below threshold): 0.01 Pearson's R value (above threshold): 0.44 Li's ICQ value: 0.252

Manders M2 (no threshold): 1.000 Manders M1 (threshold): 0.919 Manders M2 (threshold): 0.977 Channel 1 Ch a n n e l 2 Pearson‘s R = 0.44 Ch a n n e l 2 Pearson‘s R = 0.81 2D intensity histogram 256.0 x 256.0

Warning! zero-zero ratio too high - The ratio between zero-zero pixels and other pixels is larger 0.17. Maybe you should use a ROI.

% zero-zero pixels: 16.75 % saturated ch1 pixels: 8.25 % saturated ch2 pixels: 1.49

Warning! thresholds too low - The auto threshold method could not find a positive threshold. m (slope): 2.19

b (y-intercept): 2.20 b to y-max ratio: 0.01 Ch1 Max Threshold: -1.00 Ch2 Max Threshold: 1.00

Pearson's R value (no threshold): 0.91 Pearson's R value (below threshold): 0.13 Pearson's R value (above threshold): 0.91 Li's ICQ value: 0.443

Channel 1 Ch a n n e l 2 Pearson‘s R = 0.91

(12)

0 20 40 60 80 100 No Stress Granules Stress Granules

IMCD3 (Control)

IMCD3 + Hippu

IMCD3 (+ Tet) Anks6 kd

IMCD3 (+ Tet) Anks6 kd + Hippu

100 kD

50 kD

37 kD

-Tet

-

+

- ANKS6

- Tubulin

WB: anti-ANKS6

WB: anti-Tubulin

No Stress Granules

Stress Granules

Anks6

Anks6 kd

Anks6 + Hippu

Anks6 kd + Hippu

a

b

_c

Supplemental Figure 11

%

(13)

(14)

Supplemental Figure 13

a

a-Flag

no Ab

10%

input

RT + -

+ -

+

150 bp

200 bp

350 bp

100 kD

-ant

i-AN

KS6

Co

ntr

ol

- ANKS6

ant

i-AN

KS6

Co

ntr

ol

Lysates

IP

b

(15)

Supplemental Figure 13 (cont.)

c

Lysates

_{Affinity Purification}

Co

ntr

ol

BI

C

1 INV

AN

KS3

AN

KS6

BBS3

AR

L1

3B

d

ANKS6

RT + - + -

a-Flag no Ab

+

-10%

input

ANKS3

INVERSIN

a-Flag no Ab 10% input a-Flag no Ab 10% input

RT + -

+ -

+

-Co

ntr

ol

BI

C

1 INV

AN

KS3

AN

KS6

BBS3

AR

L1

3B

(16)

Supplemental Figure 13 (cont.)

e

f

F. A

N

K

S

3

F. A

N

K

S

6

F. IN

V

E

R

S

IN

F. B

IC

C

1

F. B

B

S

3

100 75 150

kD-V5.AGO2

- V5.AGO2

IP: Anti-Flag, WB: anti-V5

100 75 150

kD-- V5.AGO2

Lysates, WB: anti-V5

150 100 75 50 37 25

kD-- F.INVERSIN

- F.BICC1

- F.ANKS6

- F.ANKS3

- F.BBS3

IP: Anti-Flag, WB: anti-Flag

F. A

N

K

S

3

F. A

N

K

S

6

F. IN

V

E

R

S

IN

F. B

IC

C

1

F. B

B

S

3 CAPRIN1.GFP

100 150

kD-- CAPRIN1.GFP

100 150

kD-Lysates, WB: anti-V5

- CAPRIN1.GFP

IP: Anti-Flag, WB: anti-V5

150 100 75 50 37 25

kD-- F.INVERSIN

- F.BICC1

- F.ANKS6

- F.ANKS3

- F.BBS3

(17)

Supplemental Figure 13 (cont.)

g

100 kD

50 kD

37 kD

-Tet

-

+

- ANKS6

- Tubulin

WB: anti-ANKS6

WB: anti-Tubulin