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, researchers at Monash University 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 RNA-seq 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.
Poly(A)-Test sequencing. (A) Schematic representation of the PAT-seq approach. (B) Schematic of the experimental approach for the Crabtree Warburg metabolic shift of yeast cells transitioning from respiratory to fermentative growth. Red arrows indicate times of cell harvest; YPEG, Gal, and Glu refer to ethanol/glycerol, galactose, and glucose as carbon source. (C) The position of each adenylation site relative to the annotated transcript stop codon (0). Note the peak position for adenylation sites is ∼100 bases after the stop. The increased number of positions in the Δccr4 sample derives from loci that are silent in the wild-type strain.
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 RNA-decay 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.
PAT-seq for DGE and polyadenylation state.