Schematic representation of the key steps of the experimental workflow leading to identification of RNA processing products. (A) Experimental preparation of the cDNA library. In order to select for functional RNAs, yeast ribosomes have been used here as bait. The next important step is the size selection of ribosome-associated RNAs and the subsequent attachment of 5′- and 3′-adaptors which are marking the natural ends of the RNAs. After deep-sequencing of the library, adaptor sequences are used to select for the reads covering the full length of the original RNA molecule (both adaptors are observed). (B) Computational analysis of the data with the APART pipeline. First, reads are aligned to the reference genome and contigs together with respective coverage plots are created. Next, contigs derived from the same read sets are clustered and only non-representative contigs (marked by lighter colors) are removed from the main results list. Processing products are predicted by scanning of the coverage plots and their abundance is estimated by subtraction of the background coverage from the maximal coverage within the predicted product (abundance correspond to the area of coverage plot marked with color).

The length dependence of multiple mapping events on the level of reads and contigs observed in the ribosome-associated cDNA library. (A) Distribution of the average genomic hit numbers for reads of different lengths. No significant increase of hit numbers is observed for shorter reads. (B) Distribution of genomic uniqueness values for contigs identified in the study. Although for shorter contigs (<150 nt) higher variability of uniqness is observed, there is no strict dependence on the contig length.

A summary of the genomic features identified in the ribosome-derived cDNA library. As indicated, for most of the contigs putative processing products were observed.

Processing of a 23-mer from 5′-end of 25S ribosomal RNA. (A) The location of the detected processing product on the secondary structure diagram of large ribosomal subunit rRNA is depicted. (B) UCSC Genome Browser visualization of the APART tracks (green) within the region of contig loc.XII-464072_3929 containing the 23-mer. (C) Semi-quantitative RT–PCR with primers specific for the 23-mer using size-selected (10–50 nt) total RNA as template results in a 69-nt long PCR product. By using 10- to 50-nt long RNAs as template amplification of this 23-mer from the unprocessed full-length 25S rRNA is avoided.

Experimental validation of the APART-predicted putative processing products. (A) Processing of the tRNA-His(GUG). On the left, UCSC Genome Browser visualization of the APART tracks (green) showing two possible processing products (processing sites marked with arrows). On the right, results of the northern blot experiment using total RNA isolated from S. cerevisiae grown in different environmental conditions (lanes: 1-UV radiation, 2-anaerobic, 3-optimal, 4-high pH, 5-low pH, 6-amino acid starvation, 7-sugar starvation) with probes against 5′- and 3′-halves of the tRNA-His. Full length tRNA is marked with open arrows, processing products are indicated by filled arrows. Differential stability of both parts can be observed. (B) Processing of the tRNA-Ser(AGA) (labeling as above). The inexact ends of the contig displayed on UCSC Genome Browser visualization suggest decreased stability of the 3′-derived processing product, comparing to tRNA-His, which is reflected by the northern blot results (right). (C) Cytoplasmic localization and processing of snoRNAs. On the left, northern blot presenting subcellular localization of snoRNA 128 (identified in this study) and snoRNA13 (not found in our cDNA library). The localization of the small nuclear RNAs sn7, sn14 and sn6 in the particular cellular fractions is also shown. For the northern analysis, total RNAs prepared either from the nuclear fraction, the cytoplasmic fraction, or from the mono- or polysomal fraction were blotted. The observed northern blot signals in the polysomal samples suggest that snoRNAs are associated with translating ribosomes in yeast. On the right, identification of the processing products derived from snoRNA 128 by northern blot using total RNA isolated from yeast grown under different environmental conditions. In all panels 5S rRNA served as internal loading control.