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.

GAAGAmGAmGAGUp y2y3y7y8y9 c8c7c4c3c2c6c5 y5y6 c1c9 y1y m/z y1 c1 c3 y3 y5 c4 y8 y2 y7 y6 y9 y4 c2 c6 c8 c5 c7 c9 y2 Relative Abundance Supplementary Figure S2. CID analyses of the methylated fragment in the wild-type embryos. The CID spectrum of the A398 and A400 methylated fragment (GAAGAm 398 GAm 400 GAGUp) obtained from the wild-type embryos. The precursor ion was m/z (z= -6). The Am398 site is underlined in the fragment sequence (inset). The product ion assignments are in agreement with Fig. 3B.

Supplementary Figure S3. LC/MS analyses of rRNAs from the U26MO pr 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 U26MO pr morphants (right panels). The U26MO pr morphants show an accumulation of the A398-unmethylated fragment (A), similar to the U26MO sp 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 misMO sp or misMO pr (control MO)-injected embryos at hpf. The embryos injected with control MOs for the respective snoRNA genes are morphologically identical to the wild-type embryos. Scale bar: 500  m. U26misMO sp WT U44misMO sp U78misMO sp U26misMO pr U44misMO p r

U78MO sp WT 30 hpf Supplementary Figure S5. A close-up image of the U78 morphant head region. The U78MO sp -injected embryos show a malformed 4 th 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 (MO sp and MO sp ), control, and WT embryos. The improperly spliced gas5 transcript (237 bp including intron 10) is increased only in U44MO sp morphants, compared with the normal gas5 transcript (67 bp without intron 10) in WT and control (U44misMO pr ) embryos bp 67 bp Actin WT U44MO sp U44MO pr U44misMO pr