RT-PCR confirmation of intron fates conducted on total RNA and low-weight-enriched RNA using random priming. PCR of genomic DNA was used as positive control. (A) Agarose gel confirming ncRNAs expressed from introns and maintained in the cell. (B) Agarose gel confirming ncRNA expression accompanied by complete mRNA splicing. (C) Agarose gel confirming ncRNA expression accompanied by alternative splicing. Arrows indicate the expected size of the PCR products according to the key. Lane designations for DNA templates: G, Genomic DNA (positive control); T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. With the exception of GLC7 ncRNA, which was run on a separate gel, the images of mRNAs, introns, and ncRNAs for each gene were cropped from the same agarose gel picture with brightness and contrast applied equally across the entire image (full images available in Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, and Figure S6). For small-size PCR products, cropping included primer dimers.

Properties of loci containing intronic novel RNA predictions or snoRNAs. Quantifications were based on RNA-seq data of the same total RNA as used in Figure 2 (GEO accession no. GSE58884) and on CRAC data (GEO accession no. GSE40046). (A) Box plots showing ribosomal protein intron transcript levels in RPKM without normalization and with normalization to host gene transcript levels. Values are shown for all RP gene introns and 12 RP gene introns with predictions. (B) Association of introns with the exosome. We reanalyzed 16 independent sequencing experiments by Schneider et al. (2012) of RNA fragments cross-linked to exosome components. Reads mapping to each intron were normalized by the host gene expression estimated by RNA-seq. For each CRAC experiment an intron was given a percentile value of how frequently it was bound to an exosome component compared to other introns. Values presented are averaged percentile derived from 16 experiments and are shown for all introns, the 19 introns with predictions, and the eight introns containing snoRNAs. The levels of significance for the Mann–Whitney U-tests are represented as follows: *** P < 10⁻³; ** P < 0.01; * P < 0.05.

Characterization of the size and expression of the ncRNA within GLC7 intron. (A) Northern blot of S. cerevisiae total RNA and oligos mimicking GLC7 intron. The blots were probed with strand-specific probes showing expression of ncRNA in antisense (left panel) and sense orientation (right panel) to the GLC7 gene. The estimation of the sizes is based on the 60-nt oligos visible on the film and a comparison with the markers visible on the membrane. (B) RT-PCR on total RNA and low-weight-enriched RNA using random priming showing the expression of ncRNA within the GLC7 intron. PCR of genomic DNA was used as positive control. Names of the primers are listed on the right and are the same as indicated in C. Gel annotation: G, genomic DNA; T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. (C) Data uploaded into the University of California Santa Cruz genome browser for sequence annotation and data visualization presenting annotated GLC7 intron. Primer names used for RT-PCR are listed next to black, blue, and red boxes indicating their position with respect to the gene annotation below and the size of the corresponding PCR product. Lines joining primers mark the amplified sequences; the regions targeted for deletion by the loxP method are symbolized by the black box; the location of the strand-specific Northern probes are shown in green; and regions with the putative structure predicted by RNAz and CMfinder are shown in gray. All features map directly onto the fragment of the gene structure diagrams. The bottom panel represents the degree of conservation of gene regions among seven yeast species.