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To investigate the decay constants (half-lives) of transcript isoforms generated by alternative polyadenylation in proliferating and quiescent cells

ABSTRACT: To investigate the decay constants (half-lives) of transcript isoforms generated by alternative polyadenylation in proliferating and quiescent cells

PROVIDER: PRJNA481128 | ENA |

REPOSITORIES: ENA

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To investigate the decay constants (half-lives) of transcript isoforms generated by alternative polyadenylation in proliferating and quiescent cells

Project description:Purpose: Alternative polyadenylation (APA) can result in the generation of transcripts that terminate at different locations within the gene, which can result in different 3' UTRs. 3´ UTRs are known to contain RNA stabilizing and destabilizing motifs that provide a platform for binding of RNA binding proteins. Changes in 3´ UTRs for the different APA-generated isoforms of a gene could allow for inclusion or exclusion of these RNA stability elements. The goal of this study is determine the stability (half-lives based on transcript decay) of APA isoforms in proliferating and quiescent cells. Methods: The proliferating and quiescent (7-day contact inhibited) cells were treated with actinomycin D to stop transcription and the cells were harvested at different time points (0. 2, 4, 8 hours) for RNA isolation and global sequencing of 3´ ends. The adaptors and polyA sequenced were trimmed from each read. The reads were aligned to the human genome (hg19) using TopHat (v2.0.14). The aligned read files were used to determine the counts of APA isoforms of each gene using the code provided by Gruber et al. (PMID: 27382025). The counts of the APA isoforms were fit to an exponential decay model to obtain half-lives. The entire workflow was performed for two biological replicates: 12-1 and 12-3 strains of human dermal fibroblasts. Results: In two different fibroblast strains (12-1 and 12-3), we found that isoforms terminating at distal polyadenylation sites were more stable than isoforms terminating at proximal polyadenylation sites in quiescent, but not proliferating, fibroblasts. Conclusions: The transition from shorter to longer isoforms in quiescent cells is associated with stabilization of the transcripts.

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Global analysis of mRNA isoform half-lives: identification of stabilizing and destabilizing elements in yeast

Project description:We measured half-lives of 21,248 mRNA 3’ isoforms in yeast by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process. Interestingly, the half-lives of mRNA isoforms from the same gene, including nearly identical isoforms, often vary widely. Based on clusters of isoforms with different half-lives, we identify hundreds of sequences conferring stabilization or destabilization upon mRNAs terminating downstream. One class of stabilizing element is a polyU sequence that can interact with poly(A) tails, inhibit the association of poly(A) binding protein, and confer increased stability upon introduction into ectopic transcripts. More generally, destabilization and stabilization elements are linked to the degree to which the poly(A) tail can engage in double-stranded structures. Isoforms engineered to fold into 3’ stem-loop structures not involving the poly(A) tail exhibit even longer half-lives. We suggest that double-stranded structures at the 3’ ends are a major determinant of mRNA stability. Half-lives of 21,248 mRNA 3’ isoforms in yeast were measured by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process.

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