Fig 2: CRISPR-based screen identifies U2AF2 as a promoter of intron retention.(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. 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 CRASP-seq pipeline until Illumina paired-end sequencing of CRISPR libraries 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.

Fig 3: U2AF2 binds to intron 2-containing PURPL transcripts and promotes intron 2 retention.(A) IGV snapshot of eCLIP-seq data showing binding sites and enrichment of U2AF2 and U2AF1 on the PURPL transcripts around intron 2. eCLIP-seq data for PTBP1, PRPF8, and SRSF1 are indicated. The annotated locus by RefSeq is also indicated. eCLIP data-seq was downloaded from encodeproject.org. (B) RT-qPCR after RNA-IPs using a U2AF2 antibody with primer pairs specifically detecting PURPL transcripts as indicated in Figure S1B. U2AF2 binds to transcripts containing intron 2 but not the ones with intron 1, intron 3, or spliced exons 2 and 3. Samples were normalized to IgG-IP. 18S was used as a loading control. (C) Validation of hgRNAs using the PURPL minigene in HAP1 and RPE1 cells. The gels show RT-PCR products for PURPL transcripts with specific primers for the minigene. The hgRNAs used were targeting intergenic region, or U2AF2. The schematics next to the gel indicate the expected products of the intron-retained and spliced isoforms. (D) Western blot for U2AF2 showing successful knockdown of U2AF2 protein in SKHEP1 cells. GAPDH was used as a loading control (lower panel). (E) Schematic of the PCR primer triplet used to detect intron 2 retention (red) or splicing (purple). The length for each PCR product is indicated. (F) Gel with RT-PCR products for PURPL upon knockdown of U2AF2 with 2 different siRNAs in SKHEP1 cells. The schematics next to the gel indicate the expected products of the intron-retained and spliced isoforms. Between the two expected PCR products, we observed an extra band corresponding to the inclusion of an alternative exon inside PURPL intron 2 as observed in RefSeq, the inclusion of which is not affected by U2AF2. (G) Graph with quantitation of the gel bands in (F). Error bars represent standard deviations from 3 (G) experiments. ***p<0.001.

Fig 4: U2AF2 promotes localization of MALAT1 to nuclear speckles.(A) RNA-FISH images for MALAT1 and Immunofluorescence images for SON is shown upon transfection of SKHEP1 cells with siCTRL or siU2AF2 with two different siRNAs. MALAT1 is enriched in nuclear speckles in the siCTRL but not upon U2AF2 knockdown. (B) Quantitation of the speckle to nuclear plasma MALAT1 signal ratio in the three replicates in panel (A). ****p<0.0001.

Fig 5: Intron 2 regulates the migration capacity of MALAT1 in breast cancer cells.(A) RNA-FISH images for MALAT1 and Immunofluorescence images for SON are shown upon transfection of MDA-MB-231 cells with siCTRL or siU2AF2. MALAT1 is enriched in nuclear speckles in the siCTRL but not upon U2AF2 knockdown. (B) Quantitation of the speckle to nuclear plasma MALAT1 signal ratio in the three replicates in panel (A). (C) RNA-FISH images for MALAT1 and Immunofluorescence images for SON in MDA-MB-231 clones either WT, or whole locus MALAT1 deletion, or intron 2 deletion. When intron 2 is deleted, MALAT1 does not enrich in speckles as it is shown with SON protein. Scale bar is 10 μm. (D) Quantitation of the speckle to nuclear plasma MALAT1 signal ratio in the three replicates in panel (C). (E) Graph showing quantification of the percentage of MALAT1 transcripts enriched in speckles or being diffused in MDA-MB-231 cells in panel (C), N=2. (F) Bar graph showing the number of cells that have migrated in transwell migration assays conducted in MDA-MB-231 WT, MALAT1 KO and MALAT1 – intron 2 deletion clones. Intron 2 deletion leads to decreased migration potential. The number of cells are the sum of cells from 5 different fields, N=4. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.