Similar Datasets
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.
2023-09-25 | GSE232598 | GEO
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.
2023-09-25 | GSE232597 | GEO
Project description:Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion
| PRJNA972974 | ENA
Project description:Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion [eCLIP-seq]
| PRJNA972979 | ENA
Project description:Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion [RNA-seq]
| PRJNA972981 | ENA
Project description:The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.
| S-EPMC2442303 | biostudies-literature
Project description:SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN premRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428].
| S-EPMC5595172 | biostudies-literature
Project description:Common variants affecting mRNA splicing are typically identified though splicing quantitative trait locus (sQTL) mapping and have been shown to be enriched for GWAS signals by a similar degree to eQTLs. However, the specific splicing changes induced by these variants have been difficult to characterize, making it more complicated to analyze the effect size and direction of sQTLs, and to determine downstream splicing effects on protein structure. In this study, we catalogue sQTLs using exon percent spliced in (PSI) scores as a quantitative phenotype. PSI is an interpretable metric for identifying exon skipping events and has some advantages over other methods for quantifying splicing from short read RNA sequencing. In our set of sQTL variants, we find evidence of selective effects based on splicing effect size and effect direction, as well as exon symmetry. Additionally, we utilize AlphaFold2 to predict changes in protein structure associated with sQTLs overlapping GWAS traits, highlighting a potential new use-case for this technology for interpreting genetic effects on traits and disorders.
| S-EPMC10936842 | biostudies-literature
Project description:The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs in planta. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach's potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.
| S-EPMC10914923 | biostudies-literature
Project description:Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.
| S-EPMC2491473 | biostudies-literature