Extended Data Fig. 2: pgFARM-induced exclusion of MBNL1 exon five in multiple cell lines. | Nature Genetics

Extended Data Fig. 2: pgFARM-induced exclusion of MBNL1 exon five in multiple cell lines.

From: RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons

Extended Data Fig. 2

a, Sanger sequencing of pgFARM-edited MBNL1 exon two in HeLa/iCas9 cells. b, Long range RT-PCR analysis of MBNL1 exon two skipping (n = 1 independent experiments). c, Left, RT-PCR analysis (n = 3 biological replicates per group) of MBNL1 exon five (e5) inclusion in Cas9-expressing IMR90 cells expressing a non-targeting pgRNA (pgNTC) or pgMBNL1.a. Right, quantification of MBNL1 exon 5 inclusion. d, Left and center, RT-PCR analysis and associated quantification of Mbnl1 exon five (e5) inclusion in Cas9-expressing B16-F10 cells expressing the indicated pgRNA. Right, RT-PCR analysis (n = 3 biological replicates per group) and associated quantification of Mbnl1 exon (e5) inclusion in Cas9-expressing Melan-A cells expressing the indicated pgRNA. e, Individual Mbnl1 alleles that were cloned from gDNA of Cas9-expressing B16-F10 cells following delivery of a Mbnl1 exon five-targeting pgRNA and subjected to Sanger sequencing. f, Quantification of total MBNL1 protein levels (top) and MBNL1 protein encoded by the exon five-including isoform (bottom) before (day 0) and after (day 14) Cas9 induction in HeLa/iCas9 cells expressing the indicated pgRNA, measured by immunoblot in Fig. 1l. *, pgRNAs that induced the greatest MBNL1 exon five exclusion. Data are representative of n = 2 independent experiments. g, Scatter plot comparing pgRNA-mediated exclusion of MBNL1 exon five (e5) and inclusion of MBNL2 exon five (e5), a paralogous exon that is regulated by nuclear MBNL1. Datapoints (n = 24) are from HeLa/iCas9 cells treated with pgMBNL1.a, pgMBNL1.d, or pgMBNL1.e pgRNAs for two weeks. r, Pearson correlation; p, associated p-value computed using a two-sided Student’s t-test; shaded region, 95% confidence interval. See Source Data for uncropped gels.

Source data

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