TREND-DB-a transcriptome-wide atlas of the dynamic landscape of alternative polyadenylation

Abstract

Alternative polyadenylation (APA) profoundly expands the transcriptome complexity. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders. A major challenge in identifying mechanisms and consequences of APA (and its perturbations) lies in the complexity of RNA 3' end processing, involving poorly conserved RNA motifs and multi-component complexes consisting of far more than 50 proteins. This is further complicated in that RNA 3' end maturation is closely linked to transcription, RNA processing and even epigenetic (histone/DNA/RNA) modifications. Here, we present TREND-DB (http://shiny.imbei.uni-mainz.de:3838/trend-db), a resource cataloging the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications and further processes. TREND-DB visualizes the dynamics of transcriptome 3' end diversification (TREND) in a highly interactive manner; it provides a global APA network map and allows interrogating genes affected by specific APA-regulators and vice versa. It also permits condition-specific functional enrichment analyses of APA-affected genes, which suggest wide biological and clinical relevance across all RNAi conditions. The implementation of the UCSC Genome Browser provides additional customizable layers of gene regulation accounting for individual transcript isoforms (e.g. epigenetics, miRNA-binding sites and RNA-binding proteins). TREND-DB thereby fosters disentangling the role of APA for various biological programs, including potential disease mechanisms, and helps identify their diagnostic and therapeutic potential.

Figure 1.

Data preview of TREND-DB cataloging the dynamic landscape of alternative polyadenylation. (A) 3′ End processing of polyadenylated RNAs relies on a complex macromolecular machinery that catalyzes endonucleolytic cleavage and polyadenylation (CPA) of the nascent pre-mRNA molecule. CPA is carried out after assembly of four multi-component protein complexes (CPSF, CSTF, CFI and CFII) interacting with multiple RNA motifs. This is tightly coupled to transcription, other steps of RNA processing and even epigenetic modifications. Differential expression of individual complex components or regulation of their binding properties can redirect CPA to alternative processing sites. In addition, further mechanisms including RNA polymerase II kinetics can regulate alternative polyadenylation. (B) Global overview of alternative polyadenylation (obtained by transcriptome-wide TRENDseq) after depletion >170 components involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications, and further processes with a potential role in the definition of RNA 3′ ends. APA shortening and lengthening refer to CPA at an upstream or downstream positioned poly(A) site (PAS). This can modulate CPA at alternative PASs located in the 3′UTR (‘tandem’ APA) or in the coding sequence (CDS) and/or intronic region (‘internal’ APA), resulting in transcript isoforms with distinct regulatory properties or encoding c-terminally modified proteins. TREND-DB is designed to provide interactive and accessible exploration of this data set.

Figure 1.

Data preview of TREND-DB cataloging the dynamic landscape of alternative polyadenylation. (A) 3′ End processing of polyadenylated RNAs relies on a complex macromolecular machinery that catalyzes endonucleolytic cleavage and polyadenylation (CPA) of the nascent pre-mRNA molecule. CPA is carried out after assembly of four multi-component protein complexes (CPSF, CSTF, CFI and CFII) interacting with multiple RNA motifs. This is tightly coupled to transcription, other steps of RNA processing and even epigenetic modifications. Differential expression of individual complex components or regulation of their binding properties can redirect CPA to alternative processing sites. In addition, further mechanisms including RNA polymerase II kinetics can regulate alternative polyadenylation. (B) Global overview of alternative polyadenylation (obtained by transcriptome-wide TRENDseq) after depletion >170 components involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications, and further processes with a potential role in the definition of RNA 3′ ends. APA shortening and lengthening refer to CPA at an upstream or downstream positioned poly(A) site (PAS). This can modulate CPA at alternative PASs located in the 3′UTR (‘tandem’ APA) or in the coding sequence (CDS) and/or intronic region (‘internal’ APA), resulting in transcript isoforms with distinct regulatory properties or encoding c-terminally modified proteins. TREND-DB is designed to provide interactive and accessible exploration of this data set.

Figure 2.

Overview of the exploration workflow with TREND-DB. TRENDseq data were aligned, quantified and processed (12) to obtain the expression values of APA transcript isoforms, and the corresponding shortening index (SI) across all siRNA-conditions (displayed in the ‘Data Preview’ tab). The ‘Main View’, where an interactive APA-network map is displayed, enables users to inspect both dimensions of the underlying resource. Upon selection of a gene (left, ‘Gene View’), all siRNA conditions affecting APA of this gene are displayed, accompanied by the TRENDseq coverage plot centered on the desired region (3′UTR or gene body); genes showing a similar APA phenotype (based on the SI profiles) can also be retrieved. When selecting a siRNA-condition (right, ‘Condition View’), all APA-affected genes are listed, and users can perform functional enrichment analysis on the fly. External databases can be reached via dynamically generated action buttons, reducing the time to retrieve additional information. A dedicated instance of the UCSC Browser is provided, containing all the genomic tracks of the TRENDseq data set, with the possibility to integrate custom tracks.

Figure 3.

Selected screenshots to illustrate the exploration of the APA landscape with TREND-DB. From the ‘Main View’ tab, users can select any condition among those included in the RNAi screening, either from a dropdown menu or from the interactive APA network (A). For example, upon selection of CPSF6 (component of the CFIm protein complex, highlighted in the circle), ‘neighboring’ components/complexes that also regulate identical APA-affected target genes are highlighted. In this case, CPSF6 was found to regulate APA with a global shortening bias, as displayed by the scatterplot in (B) summarizing the PAS usage. Links to external databases (C) are automatically displayed for simplifying further exploration steps beyond TREND-DB. Moreover, a table containing the affected genes (sorted by the SI in (D)) is displayed, and can be used to further explore the APA phenotype of a particular gene, e.g. OAZ1, both in the ‘Main View’ (E), where general information is shown, and in the ‘Gene Plot’ tab, where users can inspect the coverage tracks for a locus of interest, such as the 3′UTR, where the effects of isoform shortening or lengthening are shown (F). Using the whole set of affected genes in a particular screening condition (i.e. knockdown), TREND-DB enables the direct calculation of functional enrichment of APA-affected target genes. The functional enrichment is displayed in an interactive table (G), or graphically as an enrichment map (H) for easier interpretation (for improving the readability, the single node labels have been replaced with a representative biological process for the clustered subset).