Global 3′-UTRome of porcine immature Sertoli cells altered by acute heat stress

Heat stress (HS) refers to the whole body or cells subject to high temperature for a long (chronic) or short (acute) time [1]. HS could not only induce adverse effects, such as intracellular ROS increase [2], mitochondrial damage [3], apoptosis [4], DNA damage and proliferation inhibition [5], but also modify epigenomics [6,7] and gene expression dynamics [8]. Sertoli (ST) cells are one of the two most important somatic cells located within animal testis, which can secret growth factors, lactate and hormones to provide the nutritional and molecular signaling supports for spermatogenesis [9,10]. HS treatment of boar SCs could induce oxidative damages [11]. Previously, we showed that acute HS (43 °C, 0.5 h) of in vitro cultured porcine immature ST (iST) cells could exert immediate and prolonged effects, such as reduced viability, promoted apoptosis and elevated lactate secretion, and extensively changed global transcriptome, which affected multiple signaling pathways, including metabolic and reproductive process [12].

In most eukaryotic genes, alternative polyadenylation (APA) is an important mechanism of producing transcript isoforms with different 3′-UTRs, due to the usage of variable polyadenylation sites [13,14]. Spatiotemporal programs on regulating mRNA expression at post-transcriptional level could be executed by cis-elements within 3′-UTR [15]. First, hexamer polyadenylation sites (PAS) and cytoplasmic polyadenylation elements (CPE), as important 3′-UTR elements, could regulate cytoplasmic polyadenylation, and affect the degradation and translation of mRNAs, through interacting with trans-factors of cytoplasmic polyadenylation specific factor (CPSF) and CPE binding proteins [[16], [17], [18]]. Second, multiple microRNA-targeting sites contained in 3′-UTR could be bound by microRNAs to suppress mRNA stability and abundance [19]. Third, other regulatory cis-elements contained in 3′-UTR, such as AU-rich elements (AREs), GU-rich elements (GREs) and long non-coding RNA-binding sites, could interact with RNA-binding proteins and long non-coding RNAs to affect gene expression, respectively [20,21]. Therefore, generation of 3′-UTRs of different lengths via APA events is crucial to the modulation of gene expression at the post-transcriptional layer.

High-throughput transcriptome sequencing (RNA-seq) generates huge volume of transcript expression data, sufficient for analyzing the dynamics of global 3′-UTR landscape (3′-UTRome) [18]. Dynamic analysis of alternative polyadenylation from RNA-seq (DaPars) was developed to identify de novo dynamic APAs and analyze 3′-UTR length based on read coverage between two conditions [18,22,23]. APAtrap was another tool to identify and quantify APA sites by tallying all potential poly(A) sites, based on the mean squared error model and capable of characterizing novel 3′-UTRs [14].

In the present study, based on our previously published RNA-seq datasets of pig iST cells before and after acute heat stress treatments [12], DaPars and APAtrap tools were used to recognize dynamic APA sites, and explore 3′-UTRome dynamics. Furthermore, cis-elements (PAS, CPE and microRNA binding sites) in 3′-UTRs of different transcript isoforms were also analyzed, with the aim to provide insights into functional roles of 3′-UTR dynamics during quick and delayed responses of acute heat stress on pig iST cells.

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