Project description:The goal of this study was to investigate the role of intragenic CTCF in alternative pre-mRNA splicing through a combined CTCF-ChIP-seq and RNA-seq approach. CTCF depletion led to decreased inclusion of weak upstream exons.
Project description:The goal of this study was to investigate the role of intragenic CTCF in alternative pre-mRNA splicing through a combined CTCF-ChIP-seq and RNA-seq approach. CTCF depletion led to decreased inclusion of weak upstream exons.
Project description:RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we evaluated 718 human RBPs with tethered function luciferase-based splicing reporter assays to identify 58 candidates, including known splicing factors such as RBFOX and serine-arginine proteins. We performed enhanced CLIP, RNA-seq, and affinity purification-mass spectrometry to investigate a subset of the 11 candidates with no prior association with splicing. Integrative analysis of these assays indicated the surprising roles of TRNAU1AP, SCAF8, and RTCA in modulating hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using identified domains, we engineered programmable fusion proteins which outperformed current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. Altogether, our tethering approach characterized the ability of RBPs to induce exon inclusion and yielded new molecular parts for programmable splicing control.
Project description:RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we evaluated 718 human RBPs with tethered function luciferase-based splicing reporter assays to identify 58 candidates, including known splicing factors such as RBFOX and serine-arginine proteins. We performed enhanced CLIP, RNA-seq, and affinity purification-mass spectrometry to investigate a subset of the 11 candidates with no prior association with splicing. Integrative analysis of these assays indicated the surprising roles of TRNAU1AP, SCAF8, and RTCA in modulating hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using identified domains, we engineered programmable fusion proteins which outperformed current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. Altogether, our tethering approach characterized the ability of RBPs to induce exon inclusion and yielded new molecular parts for programmable splicing control.
Project description:Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression, but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as three changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that three of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation. Keywords: Time course; Splicing-sensitive microarray For Exon array hybridizations: 3 biological replicates of day 7, 2 biological replicates of day 10, 3 biological replicates of day 14; For HJAY array hybridizations: 5 biological replicates each were analyzed from day 7 and day 14. One day 14 replicate was deemed an outlier and removed from subsequent analyses.
Project description:Intragenic 5-methylcytosine and CTCF mediate opposing affects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF binding sites were unclear. In this study, we identify the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives.
