Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: SRSF1 but not SRSF2 levels drive the amounts of full-length and D2-CD33 mRNA transcripts in opposite directions and modulate the quantity of CD33 protein at the cell surface. (A and B) Fraction of CD33 mRNA transcripts expressed as D2-CD33 in HeLa cells overexpressing SRSF1 or SRSF2, normalized to the empty vector control (A). In the same samples, mRNA levels of CD33 exon 2 included (black bars) and exon 2 skipped (gray bars) mRNA transcripts were measured by RT-qPCR (B). (C) Relative mRNA levels of SRSF2 transcripts in THP1 monocytes upon knockdown, measured by RT-qPCR compared to the NTC. (D to F) Flow cytometry analysis of CD33 protein surface levels using labeled antibodies targeting CD33 exon 2 (D) or exon 3 (E) in HeLa cells coexpressing SRSF1 or SRSF2 with a CD33 minigene. The mean fluorescence intensities (MFI) are quantified in panel F and were normalized to control cells expressing the CD33 minigene only. (G and H) Fraction of CD33 mRNA transcripts expressed as D2-CD33 upon SRSF1 or SRSF2 knockdown in THP1 monocytes, normalized to cells transfected with an NTC siRNA (G). In the same samples, NTC-normalized levels of full-length CD33 and D2-CD33 were determined by RT-qPCR (H). (I to L) Flow cytometry analysis of CD33 and CD11b protein surface levels using FITC- or PE-labeled antibodies targeting CD33 exon 2 (I) or exon 3 (J) or CD11b (K) in THP1 monocytes upon knockdown of SRSF1 or SRSF2. CD33 KO cells were utilized as a negative control. The NTC-normalized MFIs are shown in panel L. Error bars indicate means plus the SEM (A, B, F, G, H, and L) or medians + the SEM (C). A one-way ANOVA was performed for each plot. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. n = 3 biological replicates for each plot. α IgV, anti-IgV; α IgC, anti-IgC 2 domain.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Plasmid Preparation, Control, Quantitative RT-PCR, Knockdown, Flow Cytometry, Labeling, Fluorescence, Expressing, Transfection, Negative Control

Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: Disrupting a predicted SRSF1 binding site near the 3′ end of exon 2 increases CD33 exon 2 skipping. (A) Overview of the predicted SRSF1 binding sites, as well as the ASO targeting sequence in CD33 exon 2. The numbered key indicates the identity of binding/targeting site mutants used in panel B. (B and D) T-allele-normalized fraction of CD33 mRNA transcripts lacking exon 2, as measured by RT-qPCR in HeLa cells transfected with CD33 minigenes carrying mutations as shown in panel A. (C) Fold change of full-length CD33 and D2-CD33 mRNA transcripts in HeLa cells overexpressing a mutant CD33 minigene compared to WT CD33 minigene-overexpressing cells, as measured by RT-qPCR. (E and F) Flow cytometry analysis of CD33 surface levels using a labeled antibody targeting CD33 C 2 domain in HeLa cells overexpressing WT or mutant 12 containing CD33 minigenes in combination with either rs12459419 C or rs12459419 T . The MFIs were normalized to cells transfected with WT-CD33 minigenes carrying rs12459419 T . Error bars indicate means plus the SEM. A one-way ANOVA (B to D) and a two-tailed t test (F) were performed. **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant. n = 3 biological replicates for each plot.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Binding Assay, Sequencing, Quantitative RT-PCR, Transfection, Mutagenesis, Flow Cytometry, Labeling, Two Tailed Test

Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: PTBP1 shows a reduced binding to the intron-exon junction at the 5′ of exon 2 in RNA fragments carrying the rs12459419 T allele. (A) Workflow for the reverse SILAC LC-MS/MS analysis of the CD33 RNA fragment pulldown. DB, desthiobiotin; S, streptavidin. (B) Log 2 ratio of the reverse ( y axis) and forward ( x axis) SILAC experiments. Red dots indicate proteins that are >4-fold enriched in the rs12459419 C pulldown, whereas blue dots indicate proteins that are >4-fold more abundant in the rs12459419 T samples. The sizes of the circles correspond to the number of unique peptides identified. Only proteins that were identified in all four biological replicates are shown. (C to E) Fraction of CD33 mRNA transcripts expressed as D2-CD33 (C), full-length CD33 (D), and D2-CD33 (E) mRNA levels measured by RT-qPCR in HeLa cells coexpressing PTBP1, SRSF1, or MATR3 with a CD33 minigene containing either rs12459419 C or rs12459419 T , normalized to cells transfected with the wild-type T-allele minigene. (F) Fraction of CD33 mRNA transcripts expressed as D2-CD33 measured by RT-qPCR in HeLa cells expressing a CD33 minigene containing either the endogenous or an extended polypyrimidine (pY) tract in the presence of rs12459419 C or rs12459419 T , normalized to wild-type cells containing rs12459419 T . Error bars indicate means plus the SEM. A one-way ANOVA was performed. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant. n = 3 biological replicates for each plot, except for the SRSF1 isoform 1 sample in panels C and E, where n = 2 due to levels of D2-CD33 being below the limit of detection in one of the biological replicates.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Binding Assay, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR, Transfection, Expressing

Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: SRSF1 knockdown increases CD33 exon 2 skipping. (A) Overview of the splicing reporter incorporated at the endogenous CD33 locus in K562 cells (top), as well as the siRNA screening strategy to identify modifiers of CD33 exon 2 splicing (bottom). (B and C) Percent change in luminescence upon knockdown of the SRSF family members (B) or hnRNP family members (C) in the splicing reporter cell line compared to NTC-transfected cells. For each gene, three different siRNAs were tested in triplicate, except for SRSF11, hnRNPCL1, and hnRNPH2, where two different siRNAs were tested, and hnRNPCL2, where a single siRNA was tested, due to the availability of predesigned siRNAs. Red boxes mark ±35% compared to the NTC. Each dot represents a single well. Purple, green, and blue dots correspond to unique siRNA reagents. (D and E) Relative mRNA levels of SRSF1 transcripts (D) or hnRNPH1 transcripts (E) in K562 cells upon knockdown of the corresponding genes, measured by RT-qPCR compared to the NTC. (F) Percent change in luminescence upon knockdown of SRSF1 and hnRNPH1 in the splicing reporter cell line compared to NTC-transfected cells. (G and H) The fraction of total CD33 transcripts lacking exon 2 in K562 cells (G) and THP1 monocytes (H) upon knockdown of SRSF1 and/or hnRNPH1 compared to the NTC, as measured by RT-qPCR. (I) Fold change of full-length CD33 and D2-CD33 mRNA transcripts in THP1 monocytes upon knockdown of SRSF1 compared to the NTC. (J) RT-PCR products from a single primer set that simultaneously detects full-length CD33 (FL) and D2-CD33 (D2) in THP1 monocytes transfected with the NTC or upon knockdown of SRSF1, visualized on a 1.2% agarose gel. The percent D2-CD33 values ± the SEM are indicated below each lane. (K to M) Flow cytometry analysis of CD33 and CD11b surface levels using FITC- or PE-labeled antibodies targeting CD33 exon 2 (K, left) or exon 3 (K, right) or CD11b (L) in THP1 monocytes upon knockdown of SRSF1. CD33 KO cells were utilized as a negative control. The NTC normalized mean fluorescence intensity (MFI) is quantified in M. A one-way ANOVA was performed. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. NTC, nontargeting control. Error bars indicate means plus the SEM. n = 3 biological replicates for each plot.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Flow Cytometry, Labeling, Negative Control, Fluorescence, Control

Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: Treatment with a 15-nucleotide MOE-ASO that targets near the 3′ end of exon 2 increases CD33 exon 2 skipping. (A) Percent change in luminescence upon transfection of K562 splicing reporter cells with MOE-ASOs targeting the CD33 RNA. Red boxes mark ±35% compared to the NTC. (B) Percent change in luminescence from K562 splice reporter cells transfected with MOE-ASOs 8 and 79 compared to the NTC. (C and D) Fraction of CD33 mRNA transcripts lacking exon 2, based on RT-qPCR analysis in K562 cells (C) and THP1 monocytes (D) upon MOE-ASO treatment, compared to the NTC. (E) NTC-normalized expression of CD33 exon 2 present (black bars) and exon 2 absent (gray bars) mRNA transcripts in THP1 monocytes upon treatment with ASO 79. (F) RT-PCR products from a single primer set that simultaneously detects full-length CD33 (FL) and D2-CD33 (D2) in THP1 monocytes transfected with the NTC or MOE-ASO 79, visualized on a 1.2% agarose gel. The percent D2-CD33 values ± the SEM are indicated below each lane. (G to I) Flow cytometry analysis of CD33 and CD11b surface levels using FITC- or PE-labeled antibodies targeting CD33 exon 2 (G, top) or exon 3 (G, bottom) or CD11b (H) in THP1 monocytes upon knockdown of SRSF1 or treatment with MOE-ASO 79. CD33 KO cells were utilized as a negative control. α IgV domain, anti-IgV domain; α IgC domain, anti-IgC 2 domain. The NTC-normalized MFIs are quantified in panel I. A one-way ANOVA (B, C, and I) and a two-tailed t test (D and E) were performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. NTC, nontargeting control. Error bars indicate means plus the SEM. n = 3 biological replicates for each plot.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Flow Cytometry, Labeling, Knockdown, Negative Control, Two Tailed Test, Control

Journal: Molecular and Cellular Biology

Article Title: SRSF1 and PTBP1 Are trans -Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk

doi: 10.1128/MCB.00568-18

Figure Lengend Snippet: Proposed model for CD33 exon 2 splicing regulation. We propose that SRSF1 binding near the 3′ end of exon 2 enhances exon 2 inclusion in the CD33 pre-mRNA, regardless of the rs12459419 SNP genotype. At the same time, PTBP1 binding to the CD33 pre-mRNA also enhances exon 2 inclusion, potentially by antagonizing the action of other splicing regulators that functionally inhibit splice junction recognition. The rs12459419 T SNP reduces the binding of PTBP1 to the proximal Py tract, thus leading to elevated exon 2 skipping.

Article Snippet: We also used the following TaqMan assays: SRSF1, Hs00199471_m1; SRSF2, Hs01089823_m1; GAPDH, Hs02758991_g1; and hnRNPH1, Hs00800662.

Techniques: Binding Assay