LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival

Abstract

Background The sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. Results To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated. Conclusions We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

The Role of Alternative Polyadenylation in the Regulation of Subcellular RNA Localization

“…The association between distal PAS usage and localization of transcripts to projections of migrating cells was also observed in NIH3T3 and MDA-MB231 cells (Goering et al, 2021). Further, it was demonstrated that the use of distal PAS in both tandem UTR and ALE structures was associated with localization to cellular projections (Goering et al, 2021).…”

“…General trends of RNA localization to membranes and 3′ end choice have been revealed by LABRAT, an APA quantification software (Goering et al, 2021). LABRAT analysis of biochemical fractionations derived from three different cell types (Benoit Bouvrette et al, 2018) revealed that longer (more distal APA) transcripts are enriched in the membrane-associated fraction (Figure 2B) (Goering et al, 2021).…”

“…The use of distal ALEs mostly led to inclusion of more binding motifs for RNA binding proteins in the 3′ UTR, including proteins in the Muscleblind-like (MBNL) family (Taliaferro et al, 2016). The association between distal PAS usage and localization of transcripts to projections of migrating cells was also observed in NIH3T3 and MDA-MB231 cells (Goering et al, 2021). Further, it was demonstrated that the use of distal PAS in both tandem UTR and ALE structures was associated with localization to cellular projections (Goering et al, 2021).…”

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3′ processing in mouse models of pontocerebellar hypoplasia

“…4 A ). The relative usage of these APA isoforms in our RNA-seq libraries was analyzed using lightweight alignment-based reckoning of alternative three-prime ends (LABRAT) ( 21 ), which generated a ψ score (ranging from 0 to 1) for each gene in each genotype. Lower ψ values indicate preferential use of proximal PASs and higher values indicate preferential use of distal PASs.…”

“…RNA-seq reads were trimmed and pseudoaligned to the transcriptome, and differential expression was analyzed using sleuth ( 44 ). Customized reference transcripts and PACs were used by LABRAT to analyze PAS usage ( 21 ). Detailed methods are provided in the SI Appendix , Supplementary Text .…”