Fig. 5

Atypical SNORD13-related RNA guides rRNA acetylation in D. melanogaster. (A) Simplified phylogenetic tree of Arthropoda. Representative species in which SNORD13-like were identified are indicated. (B) Simplified phylogenetic tree of Diptera. The genomic arrangements of newly-identified SNORD13 genes (blue arrow) are shown. (C) Multiple sequence alignment of representative eukaryotic SNORD13 sequences. The small red arrow indicates the relative position of the mature 5′-end of Or-CD1 in Diptera (see also panel F) while the two horizontal black arrows in opposite orientation depict the terminal 5′-3′ stem structure. Note that SNORD13 sequences in Diptera - but not in other species - lack one of the two conserved antisense rRNA elements (denoted by horizontal blue bars). Vertical black, grey and white bars highlight Diptera, other Arthropoda and non-Arthropoda species, respectively. (D) Immunoprecipitation by anti-trimethyl cap (R1131) antibodies. OR-CD1 in various insects (as indicated below the panels) was detected by Northern blots. The same membrane was hybridized with an antisense oligo probe that recognized uncapped 5.8S rRNA used as negative controls. Input RNA (I); RNA recovered from the pellet (P). RNA recovered from the supernatant (S). The theoretical size of SNORD13 (nt) in each of the species studied is indicated in parentheses. (E) Schematic representation of two distinct modes of expression: independent transcription gives rise to short (top) or long (bottom) forms of 5′ capped SNORD13. (F) A mutant KO fly strain harboring an inserted P-element in the Or-CD1 gene (Dmel\P{EP}snoRNA:Or-CD1G9117) does not express OR-CD1 as assayed by primer extension. Note that the 5′-end of the cDNA product confirms that fly Or-CD1 lacks one 18S rRNA complementarity. The expected size (nt) of OR-CD1 with ∼4–5 nucleotides upstream of the C-box (as shown on the panel) is indicated in parentheses. ³²P labeled primer (P). (G) Misincorporation (C-to-U) at SSU-C1968 (helix 45) as judged by Sanger DNA sequencing of RT-PCR products obtained after borohydride treatments of total RNA extracted from WT and Or-CD1-KO adult flies.