Xenbase Image ID: 171778

Figure 5. MOs Can Cause Off-Target Mis-splicing (A) Quantitative comparison of t transcript levels and splice junction usage between t/t2 MO-injected and uninjected tailbud embryos in a superimposed Sashimi plot. Canonical and cryptic splicing are shown with solid and dashed lines, respectively. Magnification of the first intron indicates position and altered usage (see arrow after abbreviation for various splice sites) of splice junctions caused by the t donor splice-blocking MO (MOsplice): a, acceptor splice site; d, canonical donor splice site; cr1-4, cryptic splice sites. (B) Consensus sequence of all canonical donor splice junctions detected in the transcriptome of X. tropicalis tailbud embryos and best alignment with control and donor splice-blocking MOs. (C) Seriated heatmap of differentially used intron clusters, transcript level changes, and MO match length at blocked splice junctions for indicated conditions compared with their uninjected controls. Selected intron clusters represent coupled splicing events that were inversely mis-regulated in either t/t2 MO- or cMO-injected tailbud embryos: inhibition of canonical (can) splicing caused alternative or cryptic (alt/cr) splice sites to be used more frequently. Blocked splice sites, 758 in t/t2 morphants and 153 control morphants, were observed with higher occurrences of reduced transcript levels (<67%; 165:54 and 19:1, respectively) and more consecutive MO base pairing (≥10; 92:26 and 49:2, respectively) than expected (nobs:nexp). (D) Temporal dynamics of mis-splicing (solid bar) and transcript (filled bar) fold changes (log2 scale) for transcripts dtymk, abi1, bloc1s4, and t in control (cMO) and t/t2 morphants (t/t2 MO) from neurula (stage 15) to mid-tailbud (stage 26) to late tailbud stage (stage 34). Mis-splicing was quantified by qRT-PCR (n = 3, mean ± SD) using forward primers that span alternative or cryptic splice junctions (altSJ/crSJ) as shown above the bar graph for dtymk and t. Cryptic splice junction shortens first exon of t (colored blue). Changes in transcript levels were determined at indicated exons (e). See Key Resources Table for the design of qRT-PCR primers. (E) Superimposed Sashimi plot of transcript dtymk whose splicing was affected by both cMO and t/t2 MO at tailbud stage. Canonical and alternative (between exons 2 and 5) splicing are shown with solid and dashed lines, respectively. The blocked donor splice site featured partial matches of ≥8 consecutive bases with the MOsplice of t and t2 as well as the cMO. The alignments show canonical Watson-Crick (vertical bar) and non-canonical wobble (colon) base pairing between the transcript and several MOs. (F) Confirmation of the alignment-based predictions in E by injecting single MOs or tracer sulforhodamine as indicated. Fold changes (log2 scale) to the alternative splicing and transcript level of dtymk were quantified by qRT-PCR (n = 4, mean ± SD). Two-tailed t test: ∗p < 0.1; ∗∗p < 0.01. Image published in: Gentsch GE et al. (2018) © 2018 The Francis Crick Institute. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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