Sequence library comparisons reveal noncoding RNAs. RNA sequence data are shown for MRPS35, a representative protein-coding gene that hosts a snoRNA within its intron. Nonadenylated RNAs, such as mature snoRNAs, are enriched in the rRNA-depleted library relative to the poly(A)-selected library. Sequence depth is represented on a log₂ axis. (Bottom track) One of 1706 lower-confidence snoRNA predictions generated using the snoscan algorithm (22).

Polycistronic snoRNA precursors exhibit unusual splicing patterns. (A) Splicing of the snR72-78 cluster in various fungal species (phylogenetic relations on left). snoRNAs are shown in green, introns as lines, and exons as yellow boxes, with internal exons labeled by size. Cotranscription of entire clusters has been demonstrated only for S. cerevisiae (16) and N. crassa (12). (B) Nested splicing of the snR57/55/61 snoRNA cluster in C. albicans. The 5′ splice site, branch site, and 3′ splice site sequences are shown for introns 1 (pink), 2 (blue), and 3 (orange). Gray “exons” represent sequences joined with intron 1, by splicing of introns 2 and 3, to create de novo intron 1 splice sites. (Inset) Reverse transcription polymerase chain reaction products of the snoRNA host transcript from wild-type cells and those deficient for nonsense-mediated mRNA decay (upf1-Δ) or nuclear exonucleolytic decay (rrp6-Δ). We infer the order of splicing events from the observable products: Intron 3 is nearly always removed, intron 2 is removed in a subset of transcripts (lower two bands), and intron 1 only when 2 and 3 have also been removed (lower band). Patterns in decay mutants are consistent with degradation of partially spliced host transcripts in the nucleus.

snoRNA-associated introns were lost in the Saccharomyces lineage. (A) Intron prediction scores [combined 5′ splice site and branch site species-specific PWM scores (15)] for 40 C/D box snoRNA flanking sequences (± 200 nt) in seven representative hemiascomycetes. Each snoRNA (horizontal row) is labeled according to the nomenclature of S. cerevisiae (where applicable) or N. crassa (CD39) or by the predicted C. albicans rRNA modification site. Intron scores greater than 5.0 (false-positive rate <0.4%) are shaded green. Inferred intron loss events for each snoRNA, based on parsimony, are indicated on the left and correspond to labeled branches (not drawn to scale) in the phylogeny shown above and in (B). snoRNAs that were not identified or that lack sufficient flanking sequence to score are gray and labeled NA or ND, respectively. Locations of N. crassa orthologs (green for intronic, white for exonic) are derived from (12). Intron scores for reverse-complements of flanking sequences (right) are provided as a negative control. (B) Phylogenetic pattern of snoRNA-associated intron loss. The number of loss events assigned to each branch is indicated in yellow. Species: N. crassa, Y. lipolytica, Debaryomyces hansenii, C. albicans, Kluyveromyces lactis, Kluyveromyces waltii, Zygosaccharomyces rouxii, and S. cerevisiae.