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. Author manuscript; available in PMC: 2019 Dec 4.
Published in final edited form as: Nat Struct Mol Biol. 2019 Jun 3;26(6):490–500. doi: 10.1038/s41594-019-0234-x

Fig. 1. Analysis of rRNA uridylation in Dis3l2 depleted mESCs.

Fig. 1.

(a) Probing scheme for northern blot and qRT-PCR analyses in this study. Bars represent the reverse DNA oligos used as northern blot probes or qRT-PCR reverse primers. Internal probe to detect 5.8S rRNA (blue bar), and probe used to detect 3’-extended 5.8S species (7S) throughout the entire study encompassing 12 nt at 3’-end of 5.8S rRNA and 11 nt at 5’ end of ITS2 (red bar), as well as a probe used to detect 5.8S rRNA with longer extension (8S rRNA, green bar) are represented. Similar probes encompassing mature rRNA-adjacent spacer junctions were used to detect 3’-extended 18S and 28S (grey bars). (b) qRT-PCR analysis of relative uridylation for various rRNAs and their elongated species as depicted in Fig. 1a. Oligo-dA oligo was used to synthesize the cDNAs. Bars represent mean ± SEM. Individual P values are presented; two-tailed Student’s t-test (n=2 for 18S; n=3 for 5S; n=4 for pre-18S, 7SB, 8S, 28S and pre-28S; n=5 for ITS1, and 5.8S, representing biologically independent cell cultures). (c) Northern blotting of Dis3l2 control and knockout RNA samples with mature 5.8S rRNA and 7SK (as control) probes. (d) Northern blot analysis with 7S probe. Arrow points to the 7SB rRNA and the asterisk marks mature 5.8S rRNA (see main text). (e) Distribution of 5.8S rRNA genomic extension in FLAG-mutant Dis3l2 RIP (black bars) and input (grey bars). (f) Distribution of A-tail length in extended 5.8S rRNAs in RIP (blue bars) and input (grey bars). (g) Distribution of U-tail length in extended 5.8S rRNAs in RIP (red bars) and input (grey bars). The sequence of “7SB” rRNA with 15 nt extension in ITS2 after the canonical end of mature 5.8S rRNA is shown. (h) Schematic representation of 3’-ligation RACE procedure. 3’-end uridylation of 28S rRNA (i) and 5S rRNA (j) in input and FLAG-mutant Dis3l2-bound protein re-expressed in Dis3l2 knockout mESCs. No extension or adenylation was observed prior to the U-tail (data not shown). WT, wild type; Het, heterozygote; KO, knockout. Panels c and d are representative images from 2 and 3 repeats, respectively. Uncropped blot/gel images are shown in Supplementary Data Set 1. Source Date for panel b are available online.