Extensive structural differences of closely related 3’ mRNA isoforms: links to Pab1 binding and mRNA stability

Alternative polyadenylation generates numerous 3' mRNA isoforms per gene; regulation of this process is critical in development and impaired in cancer. Individual isoforms from the same gene can vary greatly in biological properties such as translational efficiency, localization, and half-life. Even closely related isoforms - including those differing by a single nt - can have different biological properties, but the underlying mechanisms are unknown. Here we show that many highly similar yeast isoforms unexpectedly exhibit extensive structural variation and differential Pab1 binding, and that these physical properties serve as a good predictor of relative isoform stability. We developed DREADS, a dimethyl sulphate (DMS)-based technique, to obtain the first transcriptome-scale structural description of individual 3' mRNA isoforms in vivo. Strikingly, near-identical yeast mRNA isoforms arising from the same gene can possess dramatically different DMS modification profiles over hundreds of nt upstream of the poly(A) tail. DREADS analyses of the same mRNAs refolded in vitro or in different species indicate that structural differences in vivo are often due to trans- acting factors. CLIP-READS, a technique we developed to measure isoform-specific binding, reveals that differences in Poly(A) binding protein (Pab1) binding to 3' ends correlate with the extent of structural variation for closely-spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-stranded character of the poly(A) tail, and low Pab1 binding is significantly associated with mRNA stability. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, and are indicative of biological function. In vivo DREADS and SHREADS: Two independent biological replicates (A and B) each of a DMS or NAI treated sample and corresponding untreated control. For the first DMS samples and controls, two technical replicates (1 and 2) of the library preparation were performed for each biological replicate, and two sequencing runs were performed to increase sequencing depth. For the NAI samples and controls, two biological replicates were perfromed, and two sequencing runs were performed to increase sequencing depth. For the SOD1 randomization experiment, two biological replicates were performed for both DMS-treated and control samples. In vitro refolded DREADS: Two independent biological replicates A and B, each with a DMS-treated sample and an untreated control. CLIP-READS: Two independent biological replicates of the triple-Myc-tagged Pab1 strain, and one with the triple-V5-tagged Pab1 strain), each with IP and input control. Cross-species experiment: Two independent biological replicates each of D. hansenii, K. lactis, and YAC-containing S. cerevisiae strains JYAC2 and JYAC7.