Project description:SF3B1, a core component of the spliceosome involved in branch point recognition and 3’ splice site selection is frequently mutated in hematopoietic malignancies. Though its associations with clinical outcomes are unclear, mice and zebra fish with conditional SF3B1 knock-in mutations develop macrocytic anemia. A hallmark of SF3B1 mutation is an increase to cryptic 3’ splice site (C3SS) usage, a finding that is recapitulated across multiple isogenic and patient cell types. Mechanisms contributing to cryptic splice site choice and the influence of mis-splicing on posttranscriptional isoform regulation in SF3B1 mutants remains unclear. Our data indicate that SF3B1 K700E mutant Nalm-6 cells carry a significantly different set of cryptic 3’ splice sites than ones utilized in wild type cells.
Project description:The branch site / 3' splice site recognition machinery is a hotspot for disease-associated mutations. A single amino acid substitution K700E in the U2 snRNP-specific protein SF3B1 is frequently associated with cancer and is particularly common among myelodysplastic syndromes. The goal of this study is to employ the U2 IP-seq strategy in examining the differences in branch site selection in cells expressing wild type SF3B1 vs those carrying the K700E mutation.
Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells. Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.
Project description:We established a mouse model of KrasG12D, Trp53-/- and Sf3b1-K700E murine pancreatic cancer to elucidate the impact of the SF3B1 mutation found in human PDAC on the KPC mouse model.
Project description:To study the impact of SF3B1 mutations on alternative splicing and the effect of H3B-8800 splicing modulator in wild type and SF3B1-mutant chronic lymphocytic leukemia cells, we established SF3B1 K700E MEC1 CLL isogenic cell line and carried out RNA deep sequencing in SF3B1 wild type and K700E MEC1 cell lines upon H3B-8800 treatment.
Project description:Recurrent somatic mutations in spliceosome factor 3b subunit 1 (SF3B1) are identified in hematopoietic cell-derived malignancies, with SF3B1-K700E being the most common one. Here we show that Treg specific expression of SF3B1-K700E (Sf3b1K700Efl/+/Foxp3YFP-Cre) results in spontaneous autoimmune phenotypes including enlarged spleen and tissue infiltration with IFN-γ-producing CD4+ T cells. CD4+ T cells from Sf3b1K700Efl/+/Foxp3YFP-Cre mice display defective Treg differentiation and inhibitory function, which is demonstrated by failed prevention of adoptive transfer colitis by Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs. Mechanically, SF3B1-K700E induces an aberrant splicing event that results in reduced expression of a cell proliferation regulator Anapc13 due to insertion a 231 bp DNA fragment to the 5’ untranslated region (UTR). Forced expression of Anapc13 gene restores the differentiation and ability of Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs to prevent adoptive transfer colitis. Our results thus highlight the impact of a hematological malignancy-associated SF3B1 mutation on immune responses and indicate a potential relationship between splicing factor mutation-dysregulated immune responses and cancer development.
Project description:The aim of this experiment was to compare the transcriptomes of SF3B1 mutant and wildtype isogenic cells at the whole cell, nuclear and cytoplasmic levels. Mutations in SF3B1 are often found in the malignant cells of patients suffering from myelodysplastic syndromes and more rarely in other cancer types. The human K-562 leukaemia cell line was modified using CRISPR/Cas9 to integrate an A>G mutation in the SF3B1 consensus coding sequence to change the 700th codon from lysine to glutamic acid (K700E). RNA-Seq was ribo-depleted and randomly primed using SMARTer® Stranded Total RNA Sample Prep Kit. Sequencing used an Illumina NextSeq.
Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.
Project description:RNA-sequencing data from HEK293T cell models: Hek293T transfected with expression vectors of SF3B1-WT, SF3B1-R625H, SF3B1-K666T, SF3B1-K700E.