Fig 2: Negative regulation of NIPA1 expression by NIPA1-SO involves FUBP1. (A) Chromatin of cultured HUVECs were pulled down using NIPA1-SO RNA probes and subjected to protein mass spectrometry analysis. (B) RNA immunoprecipitation was used to determine if the transcription factor FUBP1, identified by mass spectrometry analysis of ChIP samples, interacts with NIPA1-SO (n = 3). An anti-FUBP1 antibody was used to immunoprecipitate cellular RNA in HUVECs, and RT-PCR was used to measure the quantity of immunoprecipitated NIPA1-SO RNA as a percentage of the input RNA. Primers amplifying the GAPDH gene were used as a control for the RT-PCR and an IgG isotype antibody was used as a control for the RNA immunoprecipitation. (C) Biotin-labeled full-length (1–1451 nt) and truncated (1–303 nt, 276–1056 nt, and 960–1451 nt) NIPA1-SO sequences were prepared by the in vitro transcription method. The labeled sequences were incubated with cell lysate at 4 °C for 3 h and then with streptavidin-conjugated magnetic beads to isolate NIPA1-SO-protein complexes, followed by Western blot analysis of FUBP1. n = 3. (D) The tertiary structure of NIPA1-SO was analyzed by RNAfold, which indicates that NIPA1-SO has three functional structure areas: 1–303 nt, 276–1056 nt, and 960–1451 nt, respectively. (E) Chromatin immunoprecipitation using an anti-FUBP1 antibody, followed by PCR analysis of NIPA1, was used to determine if FUBP1 interacts with the NIPA1 gene in HUVECs (n = 3). Data are presented as a percentage of input DNA with IgG isotype immunoprecipitation and GAPDH PCR used as controls for the immunoprecipitation and PCR, respectively. (F) Western blotting was used to determine if NIPA1-SO-mediated down-regulation of NIPA1 is dependent upon FUBP1 using lentiviral overexpression with and without FUBP1 siRNA in HUVECs (n = 5). Data are presented normalized to ß-actin and the untreated control (without lentiviral transduction or siRNA transfection). *P < 0.05 by unpaired 2-tailed Student’s t-test or one-way ANOVA with Tukey’s post hoc tests.

Fig 3: Identification of Cooperating TSGs in an In vivo Loss-of-Function CRISPR-Cas9 Screen(A) Experimental schema for CRISPR-Cas9 library in vivo screen for cooperating TSGs. The library was packaged in lentivirus used to infect MCF10F cells at an MOI = 3. Cells were selected for expression of the library and implanted into NOD-SCID mammary fat pads at 1 × 106 cells per gland. Tumors were excised and sequenced for analyses.(B) H&E staining of tumors for identification of histological phenotypes: squamous (i and v), metaplastic (ii), papillary (iii and vi), and adenocarcinoma (iv).(C) Immunohistochemistry (IHC) staining of tumors for identification of specific epithelial and proliferation markers: EpCAM (i), Ki67 (ii), PR(203) (iii), PR(403) (iv), E-cadherin (v), p53 (vi). Scale bars represent 100 µm. Magnification = 203 for microscope images.(D) Pie charts representing the clonal heterogeneity and dominant contributing and/or cooperating TSGs in the tumors. Each chart represents one tumor; each slice of each pie chart represents a subclone.(E) Mutual exclusivity analysis of FUBP1 (gene A) and tumor suppressor genes that cooperated with FUBP1 in the screen (gene B) across 69,310 human cancer samples in 233 studies using cBioPortal. “Neither” represents the number of samples in which neither gene A nor B was altered. “A” represents the number of samples in which only gene A is altered. “B” represents the number of samples in which only gene B was altered. “A&B” represents the number of samples in which both genes A and B were altered.

Fig 4: Exons Upstream of FUBP1-Regulated Splice Sites Exhibit Diminished m6A Levels in FUBP1-Null Cells(A) Western blot validation of significant proteins from IP/MS experiment.(B and C) Dot blot measuring global m6A levels in mRNA of indicated cell lines (B), quantified in (C).(D) RNA-IP with m6A-modified or non-modified RNA bait followed by immunoblotting for a bona fide m6A reader, YTHDF2, and FUBP1.(E) Distribution of m6A-seq peaks across the CASP8, BRCA1, and MAGI3 loci, based on analysis of previously published m6A-seq data in HepG2 cells. The locations of the putative m6A sites are indicated within exons directly upstream of splice sites yielding AS transcripts found in FUBP1-null cells.(F) Relative m6A levels at m6A consensus sites of CASP8, BRCA1, and MAGI3 in exons upstream of splice sites that yield alternative variants, determined by m6A RIP-qPCR in NTC and FUBP1-null MCF10F cells.(G) CASP8, BRCA1, and MAGI3 mRNA levels relative to GAPDH determined by quantitative real-time PCR in NTC and FUBP1-null MCF10F cells (n = 2) using primers flanking the regions distal from splice sites, not surrounding m6A consensus sites. Data are presented as means ± SEM, n = 3 biological replicates per cell line. *p < 0.05 (two-tailed Student’s t tests), unless otherwise stated.

Fig 5: FUBP1 and Other m6A-Associated Proteins that Are Altered in Human Breast Cancers(A and B) Percentage of breast cancer samples with (A) low (left) or high (right) copy number or mRNA alterations in FUBP1 or (B) other m6A-related genes, reported by METABRIC (2,509 samples).(C and D) Schematic representation of FUBP1 mechanism in regulating alternative splicing: FUBP1 binds VIRMA and RBM15 to help recruit the rest of the m6A complex to target mRNA sites that affect splicing of cancer drivers (C). In the context of FUBP1 loss (D), there are fewer m6A modifications, thus preventing the interaction of normal m6A-binding proteins with modified sites and their downstream effects, i.e., AS of cancer driver genes.