Differentiation of syncytialized trophoblasts elicits widespread 3’UTR shortening that accompanies increased cellular function in protein secretion
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ABSTRACT: Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different 3’UTR sizes. The APA profile varies across tissue types and under different growth and differentiation conditions. Here we report that, unlike many other cell differentiation lineages, differentiation of syncytiotrophoblasts (SCTs) elicits widespread 3’UTR shortening, as shown in multiple in vitro trophoblast differentiation models as well as in single placental cells at different stages of pregnancy. We show that the 3’UTR shortening is unrelated to cell proliferation, a feature previously associated with 3’UTR size control, but rather accompanies increased cell function in protein secretion. In addition, 3’UTR shortening correlates with increased transcript abundance and is coupled with enhanced intronic APA site usage, suggesting activation of 3’ end processing in SCT differentiation. Together, our data indicate that SCTs utilize APA to express transcripts with short 3’UTRs, which may enhance hormone production and secretion, contributing to their functions in pregnancy.
Project description:Maturation of the 3¢ end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3¢ untranslated regions (UTRs). The 3¢UTR contains motifs that regulate mRNA metabolism; thus, changing the 3¢UTR length via APA can significantly impact gene expression. Endochondral ossification is a central process in bone healing, and the impact of APA on gene expression during this process is unknown. Here, we report widespread utilization of APA that impacts multiple pathways with established roles in bone healing. Importantly, progression of endochondral ossification is typified by global 3¢UTR shortening that is coupled with an increased abundance of shortened transcripts as compared to all other transcripts, underscoring the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of genes that undergo APA in the fracture callus uncover an intricate regulatory network in which APA boosts the expression of collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA does so via shortening the 3¢UTRs of Col1a1 and Col1a2 mRNAs, which removes the binding sites of miR-29a-3p that otherwise potently triggered the degradation of both transcripts. Taken together, our study takes the lead in characterizing crucial roles of APA in tailoring the 3¢UTR landscape and regulating gene expression during fracture healing.
Project description:Differentiation of syncytialized trophoblasts elicits widespread 3’UTR shortening that accompanies increased cellular function in protein secretion
Project description:Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3âUTRs in testes. Here we show widespread shortening of 3âUTR by APA during the first wave of spermatogenesis in mouse, with 3âUTRs being the shortest in spermatids. Shortening of 3âUTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear to be highly potent for transcript elimination during spermatogenesis. We additionally found widespread regulation of APA in introns and global activation of upstream antisense transcripts during spermatogenesis. Interestingly, genes that display 3âUTR shortening tend to have higher levels of H3K4me3, consistent with the open chromatin feature previously observed in spermatids. Since genes with 3âUTR shortening tend to have functions important for further sperm development after spermatids, when transcription is halted, this result indicates that expression of short, stable mRNAs may serve the purpose of mRNA storage for later translation. Thus, APA in spermatogenesis connects regulation of chromatin status with post-transcriptional control, and impacts sperm maturation. 3'READS of 1 week to 6 week of testis development
Project description:Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different lengths of 3’ untranslated region (3’UTR). Here we show arsenic stress elicits global shortening of 3’UTRs through two mechanisms. First, stress leads to immediate shortening of 3’UTR due to preferential usage of proximal cleavage and polyadenylation sites (PASs), as revealed by 3’ end sequencing of newly made RNAs that are metabolically labeled with 4-thiouridine. Second, long 3’UTR isoforms are more rapidly degraded during recovery from stress as compared to short 3’UTR isoforms, further shortening 3’UTR lengths in the cell. Using ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing (3’READS+RIP), we show that the RNA-binding protein T cell-restricted intracellular antigen-1 (Tia1) preferentially interacts with long 3’UTR isoforms via U-rich elements in alternative 3’UTR sequences, and the interaction correlates with stress granule (SG) association during stress and with mRNA decay during recovery from stress, indicating SG-mediated RNA clearance mechanism post stress. Importantly, genes whose 3’UTRs are shortened by APA during stress can evade stress-induced 3’UTR size-based mRNA degradation, leading to higher transcript abundance post stress. Moreover, proliferating and differentiated cells display different extents of 3’UTR shortening after stress, indicating cell type-specific of impact of stress on the 3’UTR landscape. Together, our data indicate that 3’UTR length plays important roles in gene expression in stressed cells, and APA functions as an adaptive stress response mechanism to preserve mRNAs.
Project description:Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’UTRs in testes. Here we show widespread shortening of 3’UTR by APA during the first wave of spermatogenesis in mouse, with 3’UTRs being the shortest in spermatids. Shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear to be highly potent for transcript elimination during spermatogenesis. We additionally found widespread regulation of APA in introns and global activation of upstream antisense transcripts during spermatogenesis. Interestingly, genes that display 3’UTR shortening tend to have higher levels of H3K4me3, consistent with the open chromatin feature previously observed in spermatids. Since genes with 3’UTR shortening tend to have functions important for further sperm development after spermatids, when transcription is halted, this result indicates that expression of short, stable mRNAs may serve the purpose of mRNA storage for later translation. Thus, APA in spermatogenesis connects regulation of chromatin status with post-transcriptional control, and impacts sperm maturation.
Project description:Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. By performing 3’RNA sequencing on acute myeloid leukemia (AML) patient samples and healthy hematopoietic stem and progenitor cells (HSPCs), we show that patient cells exhibit global 3’ untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in poly(A) site usage. Among APA regulators, FIP1L1 expression correlated with the degree of APA dysregulation and knockdown of this RNA-binding protein (RBP) reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3’UTR lengthening and downregulation of fusion oncoprotein AML1-ETO. In non-t(8;21) cells, knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study gives mechanistic insight into the role of APA in AML pathogenesis and provides evidence that targeting global APA patterns can overcome the differentiation block of AML patients.
Project description:Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. By performing 3’RNA sequencing on acute myeloid leukemia (AML) patient samples and healthy hematopoietic stem and progenitor cells (HSPCs), we show that patient cells exhibit global 3’ untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in poly(A) site usage. Among APA regulators, FIP1L1 expression correlated with the degree of APA dysregulation and knockdown of this RNA-binding protein (RBP) reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3’UTR lengthening and downregulation of fusion oncoprotein AML1-ETO. In non-t(8;21) cells, knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study gives mechanistic insight into the role of APA in AML pathogenesis and provides evidence that targeting global APA patterns can overcome the differentiation block of AML patients.
Project description:Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' UTR, introns, or exons. Most studies focus on APA within the 3' UTR, but here we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3'UTR and internal sites in a pathway-specific manner. Genes associated with neuronal function undergo mostly intronic APA, reducing their expression, while genes associated with heart and skeletal function mostly undergo 3' UTR APA and are upregulated. This suggests that, in healthy conditions, cells toggle between internal and 3'UTR APA to modulate protein expression.
Project description:About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1.
Project description:About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1.