U25

sno

RN

A

U26

sno

RN

A

U27

sno

RN

A

U28

sno

RN

A

U29

sno

RN

A

U30

sno

RN

A

U31

sno

RN

A

U22

sno

RN

A

Human

Zebrafish 22

2225

25

26

26

27

27

28 29

U22

sno

RN

A

29

22 30 31

313131 30 30

Human

Zebrafish 78

4774

74

75

76

76

79

77 78

80

44 79 80

474775 44

81

75 79 80

3125 28 22 2927 30Frog 26 31

25 27 30 3127 31Fugu 26 2229

4774 75 76 7844 79 80 81

4774 75 76 8080 44

Mouse

Fugu 79

A

B

U74

sno

RN

A

U75

sno

RN

A

U76

sno

RN

A

U77

sno

RN

A

U78

sno

RN

A

U79

sno

RN

A

U80

sno

RN

A

U47

sno

RN

A

U44

sno

RN

A

U81

sno

RN

A

Supplementary Figure S1. The exon-intron structure of the U22HG and GAS5 genes. (A) A comparative analysis of the U22HG gene in humans, frog, fugu, and zebrafish. The analyses indicate that there is a relatively high syntenic conservation, although the type and copy number of the snoRNA genes vary among these species. (B) A gene structure comparison of GAS5 in humans, mouse, fugu, and zebrafish, which indicates that there is a high degree of snoRNA gene conservation among these species.

GAAGAmGAmGAGUpy2y3y7y8y9

c8c7c4c3c2 c6c5

y5y6

c1 c9

y1y4

200 400 600 800 1000m/z

0

20

40

60

80

100

y1

c1

c3y3

y5c4

y8

y2

y7y6

y9y4

c2

c6

c8c5

c7

c9

y2

Rela

tive

Abun

danc

e

Supplementary Figure S2. CID analyses of the methylated fragment in the wild-type embryos.The CID spectrum of the A398 and A400 methylated fragment (GAAGAm398GAm400GAGUp) obtained from the wild-type embryos. The precursor ion was m/z 619.4 (z= -6). The Am398 site is underlined in the fragment sequence (inset). The product ion assignments are in agreement with Fig. 3B.

WT U26MOpr

A

B

CR

elat

ive

Abu

ndan

ce

0

100

22 32 37 42270

100

22 32 37 4227

26 30 34 38 26 30 34 38

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

0

100

0

100

0

100

26 30 34 380

100

26 30 34 38

minmin

min min

min min

GAAGAmGAmGAGUp(U26)MW 3722.567 m/z 1239.848 (z = -3)GAAGAGAmGAGUpMW 3708.551 m/z 1235.176 (z = -3)

CAAUAACAGmGp(U25)MW 3283.491 m/z 1640.738 (z = -2)CAAUAACAGpMW 2924.428 m/z 1461.206 (z = -2)

GGGGmAAAGAAGACp(HBII-99)MW 4381.667m/z 729.270 (z = -6)GGGGAAAGAAGACpMW 4367.652 m/z 726.934 (z = -6)

28S rRNA 394-404

28S rRNA 3875-3887

18S rRNA 1482-1490/1491

Supplementary Figure S3. LC/MS analyses of rRNAs from the U26MOpr morphants. Mass chromatograms of RNase A-digested 28S rRNA (A, B) or RNase T1-digested 18S rRNA fragments (C) from the wild-type embryos (left panels) and the U26MOpr morphants (right panels). The U26MOpr morphants show an accumulation of the A398-unmethylated fragment (A), similar to the U26MOsp morphants (Fig. 2A), whereas analyses of other snoRNA-guided modification sites (B, C) did not show any accumulation of unmethylated fragments in these morphants. The representations here are the same as in Fig. 2.

Supplementary Figure S4. Morphology of the mismatch MO-injected embryos. A lateral view of the wild-type and misMOsp or misMOpr (control MO)-injected embryos at 25-27 hpf. The embryos injected with control MOs for the respective snoRNA genes are morphologically identical to the wild-type embryos. Scale bar: 500 m.

U26misMOsp

WT

U44misMOsp

U78misMOsp

U26misMOpr

U44misMOpr

U78MOspWT

30 h

pf

Supplementary Figure S5. A close-up image of the U78 morphant head region.The U78MOsp-injected embryos show a malformed 4th ventricle (curved line), but do not have any other obvious defects in other parts of the brain compared with the wild-type embryos. Scale bar: 200 m.

Supplementary Figure S6. Semi-quantitative RT-PCR analysis of gas5 transcript level relative to actin in U44MO (MOsp and MOsp), control, and WT embryos. The improperly spliced gas5 transcript (237 bp including intron 10) is increased only in U44MOsp morphants, compared with the normal gas5 transcript (67 bp without intron 10) in WT and control (U44misMOpr) embryos.

1011

10 1144237 bp

67 bp

Actin

WT

U44M

Osp

U44M

Opr

U44m

isMOpr