supplementary figure s1. the exon-intron structure of the u22hg and gas5 genes. (a) a comparative...
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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
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y1
c1
c3y3
y5c4
y8
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y7y6
y9y4
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c8c5
c7
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Rela
tive
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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
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ive
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22 32 37 42270
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22 32 37 4227
26 30 34 38 26 30 34 38
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ativ
e A
bund
ance
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26 30 34 380
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minmin
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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
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isMOpr