Alternative polyadenylation analysis in animals and plants: newly developed strategies for profiling, processing and validation

Abstract

Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both animals and plants. Here we review newly developed next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation. The library construction methods LITE-Seq (Low-Input 3'-Terminal sequencing) and PAC-seq (PolyA Click sequencing) tag polyA+ cDNA, while BAT-seq (BArcoded, three-prime specific sequencing) and PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3′End Retrieval by CrossLinking ImmunoPrecipitation) enrich polyA+ RNA. Interestingly, only WTTS-seq (Whole Transcriptome Termini Site sequencing) targets both polyA+ RNA and polyA+ cDNA. Varieties of bioinformatics pipelines are well established to pursue read quality control, mapping, clustering, characterization and pathway analysis. The RHAPA (RNase H alternative polyadenylation assay) and 3'RACE-seq (3' rapid amplification of cDNA end sequencing) methods directly validate APA sites, while WTSS-seq (whole transcriptome start site sequencing), RNA-seq (RNA sequencing) and public APA databases can serve as indirect validation methods. We hope that these tools, pipelines and resources trigger huge waves of interest in the research community to investigate APA events underlying physiological, pathological and psychological changes and thus understand the information transfer events from genome to phenome relevant to economically important traits in both animals and plants.

A Tissue-Specific Landscape of Alternative Polyadenylation, lncRNAs, TFs, and Gene Co-expression Networks in Liriodendron chinense

“…Alternative polyadenylation is a critical post-transcriptional regulatory mechanism involved in maintaining RNA stability, ensuring accurate RNA localization and translation; these stabilities are crucial in plant development and flowering (Liu et al, 2010;Shen et al, 2011;Zhang et al, 2018). In A. thaliana, approximately 60% of the genes have multiple poly(A) sites (Shen et al, 2011).…”

Extensive Involvement of Alternative Polyadenylation in Single-Nucleus Neurons

“…During past few years, a growing number of APA profiles have been examined across multiple tissues for mammal species, using next-generation sequencing methods and technologies to capture the 3'-end of transcripts [20][21][22]. However, these NGS-based methods generate bulk genome or transcriptome data, which cannot provide a high-resolution view of cell-to-cell variation.…”

DPAC: A Tool for Differential Poly(A)–Cluster Usage from Poly(A)–Targeted RNAseq Data

“…Nonetheless, the DPAC pipeline is applicable to any data type provided that there are poly(A) tracts (or poly(T) tracts in the negative sense) retained within the read data that are of at least 10nts in length and the read length after poly(A) trimming is greater than 25nts. There are many current poly(A)-tail focused methods for RNAseq (Zhang et al 2018), that yield similar read data focused on the 3′UTR and poly(A)-tail junction. So to demonstrate this functionality, we ran the DPAC pipeline using previously deposited and published datasets to generate de novo PAC datasets: 1) 3′READs+ data derived from HeLa cells (human) (Zheng et al 2016) and 2) PAS-Seq datasets derived from MEF cells (murine) (Chang et al 2018).…”