Figure 6. Validation of alternative 5’-end sequences associated with some snoRNAs.

A) Primer extension of RNA derived from either of two different wild-type S. cerevisiae strains (BY4743 or SC126) to corroborate 5’-end heterogeneity in yeast snoRNAs suggested by RNAseq reads. The colored bars shown above each panel correspond to possible 5’-end sequences of each snoRNA, with the annotated 5’-end (+1) indicated in white boxes with orange outline. Sequences corresponding to additional nucleotides present at the 5’-end (−3, −2, −1) are indicated in red, gray, and blue respectively. Sequences corresponding to shorter RNA 5’-ends (+2, +3) are indicated by purple and green, respectively. For longer RNA 5’-ends, if no reads with this 5’-end sequence were observed by RNAseq, the identity of the nucleotide is indicated by X, since the primer extension experiment applied here in the presence of all four dNTPs only indicates the length of the RNA, and not sequence. The first lane (P) in each panel corresponds to the primer only control reaction, and the three subsequent lanes represent extension of the RNA with varied dNTP (400, 150, 50 μM) to rule out additional nucleotides added by RT at high concentration of dNTP. Lanes A,T,G,C correspond to ddNTP sequencing lanes. Colored arrows are used to mark the positions of abundant primer extension products corresponding to each RNA 5’-end, as indicated above the panel. B) Quantification of percent of total primer extension products corresponding to each 5’-end stop position for the indicated RNAs from each strain. Extension results were quantified using the reactions containing 150 μM dNTP (middle concentration of the three extension lanes) and bars are colored to indicate the 5’-end that is represented using the same colors as in panel A. The most abundant 5’-end that was observed in the RNA-seq read data is indicated below each RNA for comparison to the primer extension results.