OncoSplicing 3.0: an updated database for identifying RBPs regulating alternative splicing events in cancers

Abstract

Alternative splicing (AS) is a crucial mechanism to regulate gene expression and protein complexity. RNA-binding proteins (RBPs) play an important role in regulating abnormal alternative splicing in cancers. However, few resources are available to identify specific RBPs responsible for regulating individual AS event. We have developed the OncoSplicing database for integrative analysis of clinically relevant alternative splicing events in TCGA cancers. Here, we further updated the OncoSplicing database by performing correlation analysis between the splicing and mRNA expression data from the TCGA cancers or GTEx tissues, mapping known RNA-binding motifs and eCLIP-seq peaks to all AS events, conducting splicing analysis for RNA-seq data from RBP perturbation experiments in the ENCODE project, and integrating exon and intron sequences for each AS event. With this updated database, users can easily identify potential RBPs responsible for the queried AS event and obtain sequences to design AS-specific primers and minigene constructs for experiment validation. Overall, compared to the previous version, the substantially updated OncoSplicing database (www.oncosplicing.com) offers a more valuable resource for users to identify RBPs responsible for regulating alternative splicing events in cancers.

Graphical Abstract

Figure 1.

Data processing and database construction pipeline. (A) Collection of AS events based on the TCGA SpliceSeq and SplAdder project. (B) Mapping RNA-binding motifs and eCLIP-seq peaks to the collected AS events, processing RNA-seq data from RBP perturbation experiments and performing correlation analyses to identify potential AS event-RBP pairs. (C) Integration of processed results into three modules: ‘MapAS’, ‘Encode’ and ‘CoExp’ for data exploration and visualization.

Figure 2.

Overview of the updated OncoSplicing database. (A, B) Browser bar in the updated OncoSplicing and an example of search results on the ‘MapAS’ page. After querying AS event ‘exon_skip_497 057’, a result table response with detailed information of the query AS event, summary results and function buttons. (C) Tracks plot displayed the relative location of motifs or peaks of SRSF1 to the structured AS event. (D) Sequences of AS events obtained through ‘Seq’ function in the ‘MapAS’ page. Sequences of flanking exons can be used to design AS specific primers (1 and 2). Sequences including and flanking the alternate exon can be used to design minigene construct (3). RNA binding motifs of SRSF1 were highlighted in red, which can be used to design site-mutation minigene constructs. (E) Tracks and Sashimi plot showed signal differences between normal control and perturbation of SRSF1 samples. (F) Volcano plot identified RBPs that affected alternative splicing of SYK after perturbation. (G) Volcano plot showed all regulated AS events after the perturbation of SRSF1 in the K562 cell line. (H) Scatter plot displayed the correlation result between the PSI value of ‘exon_skip_497 057’ and mRNA expression of ESRP1 in the TCGA pancreatic adenocarcinoma (PAAD) cohort.