PAT-seq: A method to study the integration of 3′-UTR dynamics with gene expression in the eukaryotic transcriptome

Abstract

A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3′ focused RNAseq methods in that it depends strictly on 3′ adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNAdecay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3′-UTR dynamics genome wide.