Journal: bioRxiv
Article Title: Non-canonical function of the splicing activator U2AF2 in promoting intron retention in the lncRNAs PURPL and MALAT1
doi: 10.64898/2026.02.19.706780
Figure Lengend Snippet: (A) Schematic of the PURPL IR reporter used in the screen to identify IR regulators. The pLCHKO vector contains the puromycin resistance gene used for selection. The transgene also contains the genomic sequences of exon 2 (161 bp), intron 2 (1,943 bp), and exon 3 (163 bp) of the PURPL gene under a Doxycycline-inducible promoter. A hybrid guide (hg)RNA for guiding SpCas9 and LbCas12a is expressed under a U6 promoter. A construct with a hgRNA targeting an intergenic region was used as a negative control. Top arrow: The hgRNA is cleaved by Cas12a to produce 2 guide RNAs for dual targeting of each protein coding gene, paralog targeting, and genetic interaction mapping. Bottom arrow: Upon intron splicing or intron retention, two different reads are generated. (B) Two different cell lines, stably expressing Cas9 and Cas12a, are used: HAP1, and RPE1. The pipeline before RNA-seq is depicted. (C) Heatmap showing single genes only (after removing gene pairs), whether identified individually or as part of a gene pair. The hits are ordered by the average ΔPIR values across HAP1 and RPE1, from lowest to highest. (D) Left: Gene ontology enrichment analysis of biological processes regulated by the hits of the screen. The most significant pathways involve mRNA processing and RNA splicing. Right: Gene ontology enrichment analysis of cellular components regulated by the hits of the screen. These components include mainly Spliceosomal Complex, small nuclear ribonucleoprotein complex and the Catalytic step 2 Spliceosome. (E) Venn diagram showing that there are 5 common hits which cause a reduction in the percentage of intron retention (ΔPIR) of the splicing reporter in the two cell lines. (F) Table showing the top 5 hits of the screen with a ΔPIR for each hit in each cell line and their full names.
Article Snippet: For HCT116 MALAT1 knockout, we used integration by non-homologous end joining, which was accomplished by introducing a simultaneous double-strand break in genomic DNA and in the targeting vectors (pCMV-Puro and pCMV-Hygro)., Plasmids encoding spCas9 and sgRNAs were obtained from Addgene (Plasmids #41815 and #47108).
Techniques: Plasmid Preparation, Selection, Genomic Sequencing, Construct, Negative Control, Generated, Stable Transfection, Expressing, RNA Sequencing
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