Alternative Polyadenylation Dynamics Research Articles

Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis.

Alternative polyadenylation (APA) is an essential post-transcriptional process that produces mature mRNA isoforms by regulating the usage of polyadenylation sites (PASs). APA is involved in lymphocyte activation; however, its role throughout the entire differentiation trajectory remains elusive. Here, we analyzed single-cell 3'-end transcriptome data from healthy subjects to construct a dynamic-APA landscape from hematopoietic stem and progenitor cells (HSPCs) to terminally differentiated lymphocytes. This analysis covered 19973 cells of 12 clusters from five lineages (B cells, CD4+ T cells, CD8+ T cells, natural killer cells, and plasmacytoid dendritic cells). A total of 2364 genes exhibited differential 3'UTR PAS usage, and 3021 genes displayed differential intronic cleavage during lymphoid differentiation. We observed a global trend of 3'UTR shortening during lymphoid differentiation. Nevertheless, specific events of both 3'UTR shortening and lengthening were also identified within each cluster. The APA patterns delineated three differentiation stages: HSPCs, precursor cells, and mature cells. Moreover, we demonstrated that the conversion of naïve T cells to memory T cells was accompanied by dynamic APA in transcription factor-encoding genes (TCF7 and NFATC2IP), immune function-related genes (BCL2, CD5, CD28, GOLT1B, and TMEM59), and protein ubiquitination-related genes (UBE2G1, YPEL5, and SUMO3). These findings expand our understanding of the underlying molecular mechanisms of APA and facilitate studies on the regulatory role of APA in lymphoid hematopoiesis.

Dynamic Alternative Polyadenylation during Litopenaeus Vannamei Metamorphosis Development.

As an important mechanism in the post-transcriptional regulation of eukaryotic gene expression, alternative polyadenylation (APA) plays a key role in biological processes such as cell proliferation and differentiation. However, the role and dynamic pattern of APA during Litopenaeus vannamei metamorphosis are poorly understood. Here, RNA-seq data covering from the embryo to the maturation (16 time points) of L. vannamei were utilized. We identified 247 differentially expressed APA events between early and adult stages, and through fuzzy mean clustering analysis, we discovered five dynamic APA patterns. Among them, the gradual elongation of the 3'UTR is the major APA pattern that changes over time, and its genes are enriched in the pathways of protein and energy metabolism. Finally, we constructed mRNA-miRNA and PPI networks and detected several central miRNAs that may regulate L. vannamei development. Our results revealed the complex APA mechanisms in L. vannamei metamorphosis, shedding new light on post-transcriptional regulation of crustacean metamorphosis.

VizAPA: visualizing dynamics of alternative polyadenylation from bulk and single-cell data.

Alternative polyadenylation (APA) is a widespread post-transcriptional regulatory mechanism across all eukaryotes. With the accumulation of genome-wide APA sites, especially those with single-cell resolution, it is imperative to develop easy-to-use visualization tools to guide APA analysis. We developed an R package called vizAPA for visualizing APA dynamics from bulk and single-cell data. vizAPA implements unified data structures for APA data and genome annotations. vizAPA also enables identification of genes with differential APA usage across biological samples and/or cell types. vizAPA provides four unique modules for extensively visualizing APA dynamics across biological samples and at the single-cell level. vizAPA could serve as a plugin in many routine APA analysis pipelines to augment studies for APA dynamics. https://github.com/BMILAB/vizAPA.

Downregulation of CPSF6 leads to global mRNA 3' UTR shortening and enhanced antiviral immune responses.

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity.

PolyAMiner-Bulk is a deep learning-based algorithm that decodes alternative polyadenylation dynamics from bulk RNA-seq data

Alternative polyadenylation (APA) is a key post-transcriptional regulatory mechanism; yet, its regulation and impact on human diseases remain understudied. Existing bulk RNA sequencing (RNA-seq)-based APA methods predominantly rely on predefined annotations, severely impacting their ability to decode novel tissue- and disease-specific APA changes. Furthermore, they only account for the most proximal and distal cleavage and polyadenylation sites (C/PASs). Deconvoluting overlapping C/PASs and the inherent noisy 3' UTR coverage in bulk RNA-seq data pose additional challenges. To overcome these limitations, we introduce PolyAMiner-Bulk, an attention-based deep learning algorithm that accurately recapitulates C/PAS sequence grammar, resolves overlapping C/PASs, captures non-proximal-to-distal APA changes, and generates visualizations to illustrate APA dynamics. Evaluation on multiple datasets strongly evinces the performance merit of PolyAMiner-Bulk, accurately identifying more APA changes compared with other methods. With the growing importance of APA and the abundance of bulk RNA-seq data, PolyAMiner-Bulk establishes a robust paradigm of APA analysis.

Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.

Single-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3' ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response. In this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3' UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile. We comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types. This study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

NUDT21 alters glioma migration through differential alternative polyadenylation of LAMC1.

Gliomas and their surrounding microenvironment constantly interact to promote tumorigenicity, yet the underlying posttranscriptional regulatory mechanisms that govern this interplay are poorly understood. Utilizing our established PAC-seq approach and PolyAMiner bioinformatic analysis pipeline, we deciphered the NUDT21-mediated differential APA dynamics in glioma cells. We identified LAMC1 as a critical NUDT21 alternative polyadenylation (APA) target, common in several core glioma-driving signaling pathways. qRT-PCR analysis confirmed that NUDT21-knockdown in glioma cells results in the preferred usage of the proximal polyA signal (PAS) of LAMC1. Functional studies revealed that NUDT21-knockdown-induced 3'UTR shortening of LAMC1 is sufficient to cause translational gain, as LAMC1 protein is upregulated in these cells compared to their respective controls. We demonstrate that 3'UTR shortening of LAMC1 after NUDT21 knockdown removes binding sites for miR-124/506, thereby relieving potent miRNA-based repression of LAMC1 expression. Remarkably, we report that the knockdown of NUDT21 significantly promoted glioma cell migration and that co-depletion of LAMC1 with NUDT21 abolished this effect. Lastly, we observed that LAMC1 3'UTR shortening predicts poor prognosis of low-grade glioma patients from The Cancer Genome Atlas. This study identifies NUDT21 as a core alternative polyadenylation factor that regulates the tumor microenvironment through differential APA and loss of miR-124/506 inhibition of LAMC1. Knockdown of NUDT21 in GBM cells mediates 3'UTR shortening of LAMC1, contributing to an increase in LAMC1, increased glioma cell migration/invasion, and a poor prognosis.

Genome-wide alternative polyadenylation dynamics underlying plant growth retardant-induced dwarfing of pomegranate.

Dwarfed stature is a desired agronomic trait for pomegranate (Punica granatum L.), with its advantages such as lower cost and increased yield. A comprehensive understanding of regulatory mechanisms underlying the growth repression would provide a genetic foundation to molecular-assisted dwarfing cultivation of pomegranate. Our previous study induced dwarfed pomegranate seedlings via exogenous application of plant growth retardants (PGRs) and highlighted the important roles of differential expression of plant growth-related genes in eliciting the dwarfed phenotype of pomegranate. Alternative polyadenylation (APA) is an important post-transcriptional mechanism and has been demonstrated to act as a key regulator in plant growth and development. However, no attention has been paid to the role of APA in PGR-induced dwarfing in pomegranate. In this study, we characterized and compared APA-mediated regulation events underlying PGR-induced treatments and normal growth condition. Genome-wide alterations in the usage of poly(A) sites were elicited by PGR treatments, and these changes were involved in modulating the growth and development of pomegranate seedlings. Importantly, ample specificities were observed in APA dynamics among the different PGR treatments, which mirrors their distinct nature. Despite the asynchrony between APA events and differential gene expression, APA was found to regulate transcriptome via influencing microRNA (miRNA)-mediated mRNA cleavage or translation inhibition. A global preference for lengthening of 3' untranslated regions (3' UTRs) was observed under PGR treatments, which was likely to host more miRNA target sites in 3' UTRs and thus suppress the expression of the corresponding genes, especially those associated with developmental growth, lateral root branching, and maintenance of shoot apical meristem. Together, these results highlighted the key role of APA-mediated regulations in fine-tuning the PGR-induced dwarfed stature of pomegranate, which provides new insights into the genetic basis underlying the growth and development of pomegranate.

Alternative 3'-untranslated regions regulate high-salt tolerance of Spartina alterniflora.

High-salt stress continues to challenge the growth and survival of many plants. Alternative polyadenylation (APA) produces mRNAs with different 3'-untranslated regions (3' UTRs) to regulate gene expression at the post-transcriptional level. However, the roles of alternative 3' UTRs in response to salt stress remain elusive. Here, we report the function of alternative 3' UTRs in response to high-salt stress in S. alterniflora (Spartina alterniflora), a monocotyledonous halophyte tolerant of high-salt environments. We found that high-salt stress induced global APA dynamics, and ∼42% of APA genes responded to salt stress. High-salt stress led to 3' UTR lengthening of 207 transcripts through increasing the usage of distal poly(A) sites. Transcripts with alternative 3' UTRs were mainly enriched in salt stress-related ion transporters. Alternative 3' UTRs of HIGH-AFFINITY K+ TRANSPORTER 1 (SaHKT1) increased RNA stability and protein synthesis in vivo. Regulatory AU-rich elements were identified in alternative 3' UTRs, boosting the protein level of SaHKT1. RNAi-knock-down experiments revealed that the biogenesis of 3' UTR lengthening in SaHKT1 was controlled by the poly(A) factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (SaCPSF30). Over-expression of SaHKT1 with an alternative 3' UTR in rice (Oryza sativa) protoplasts increased mRNA accumulation of salt-tolerance genes in an AU-rich element-dependent manner. These results suggest that mRNA 3' UTR lengthening is a potential mechanism in response to high-salt stress. These results also reveal complex regulatory roles of alternative 3' UTRs coupling APA and regulatory elements at the post-transcriptional level in plants.

StAPAminer: Mining Spatial Patterns of Alternative Polyadenylation for Spatially Resolved Transcriptomic Studies

Alternative polyadenylation (APA) contributes to transcriptome complexity and gene expression regulation and has been implicated in various cellular processes and diseases. Single-cell RNA sequencing (scRNA-seq) has enabled the profiling of APA at the single-cell level; however, the spatial information of cells is not preserved in scRNA-seq. Alternatively, spatial transcriptomics (ST) technologies provide opportunities to decipher the spatial context of the transcriptomic landscape. Pioneering studies have revealed potential spatially variable genes and/or splice isoforms; however, the pattern of APA usage in spatial contexts remains unappreciated. In this study, we developed a toolkit called stAPAminer for mining spatial patterns of APA from spatially barcoded ST data. APA sites were identified and quantified from the ST data. In particular, an imputation model based on the k-nearest neighbors algorithm was designed to recover APA signals, and then APA genes with spatial patterns of APA usage variation were identified. By analyzing well-established ST data of the mouse olfactory bulb (MOB), we presented a detailed view of spatial APA usage across morphological layers of the MOB. We compiled a comprehensive list of genes with spatial APA dynamics and obtained several major spatial expression patterns that represent spatial APA dynamics in different morphological layers. By extending this analysis to two additional replicates of the MOB ST data, we observed that the spatial APA patterns of several genes were reproducible among replicates. stAPAminer employs the power of ST to explore the transcriptional atlas of spatial APA patterns with spatial resolution. This toolkit is available at https://github.com/BMILAB/stAPAminer and https://ngdc.cncb.ac.cn/biocode/tools/BT007320.

Comprehensive mapping of alternative polyadenylation site usage and its dynamics at single-cell resolution

Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at the single-cell level is largely unexplored. Here, we developed single-cell polyadenylation sequencing, a strand-specific approach for sequencing the 3' end of transcripts, to investigate the landscape of APA at the single-cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle genes were significantly enriched in genes with high variation in polyA site usages. Furthermore, the 414 genes showing a polyA site usage switch after cell synchronization enriched cell cycle genes, while the differentially expressed genes after cell synchronization did not enrich cell cycle genes. We further identified 812 genes showing polyA site usage changes between neighboring cell cycles, which were grouped into six clusters, with cell phase-specific functional categories enriched in each cluster. Deletion of one polyA site in MSL1 and SCCPDH results in slower and faster cell cycle progression, respectively, supporting polyA site usage switch played an important role in cell cycle. These results indicate that APA is an important layer for cell cycle regulation.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA invivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the 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 provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

ScAPAmod: Profiling Alternative Polyadenylation Modalities in Single Cells from Single-Cell RNA-Seq Data.

Alternative polyadenylation (APA) is a key layer of gene expression regulation, and APA choice is finely modulated in cells. Advances in single-cell RNA-seq (scRNA-seq) have provided unprecedented opportunities to study APA in cell populations. However, existing studies that investigated APA in single cells were either confined to a few cells or focused on profiling APA dynamics between cell types or identifying APA sites. The diversity and pattern of APA usages on a genomic scale in single cells remains unappreciated. Here, we proposed an analysis framework based on a Gaussian mixture model, scAPAmod, to identify patterns of APA usage from homogeneous or heterogeneous cell populations at the single-cell level. We systematically evaluated the performance of scAPAmod using simulated data and scRNA-seq data. The results show that scAPAmod can accurately identify different patterns of APA usages at the single-cell level. We analyzed the dynamic changes in the pattern of APA usage using scAPAmod in different cell differentiation and developmental stages during mouse spermatogenesis and found that even the same gene has different patterns of APA usages in different differentiation stages. The preference of patterns of usages of APA sites in different genomic regions was also analyzed. We found that patterns of APA usages of the same gene in 3′ UTRs (3′ untranslated region) and non-3′ UTRs are different. Moreover, we analyzed cell-type-specific APA usage patterns and changes in patterns of APA usages across cell types. Different from the conventional analysis of single-cell heterogeneity based on gene expression profiling, this study profiled the heterogeneous pattern of APA isoforms, which contributes to revealing the heterogeneity of single-cell gene expression with higher resolution.

Dynamic alternative polyadenylation during iPSC differentiation into cardiomyocytes

Alternative polyadenylation (APA) is an important post-transcription regulatory mechanism widely occurring in eukaryotes and has been associated with special traits/diseases by several studies. However, the dynamic roles and patterns of APA in cell differentiation remain largely unknown. Here, we systematically characterized the APA profiles during the differentiation of induced pluripotent stem cells (iPSCs) to cardiomyocytes by the previously published RNA-seq data across 16 time points. We identified 950 differential APA events and found five dynamic APA patterns with fuzzy c-means clustering analysis. Among them, 3′UTR progressive lengthening is the main APA pattern over time, the genes of which are enriched in cell cycle and mRNA metabolic process pathways. By constructing the linear mixed-effects model, we also indicated that TIA1 plays an important role in regulating APA events with this pattern, including genes essential to cardiac function. Additionally, APA and polyA machinery activity with another pattern can immediately respond to developmental signal-mediated stimuli at the early differentiation stage and result in a sharp shortening of the 3′UTR. Finally, a miRNA-APA network is constructed and several hub miRNAs potentially regulating cardiomyocyte differentiation are detected. Our results show the complex APA mechanisms during the differentiation of iPSCs into cardiomyocytes and provide further insights for the understanding of APA regulation and cell differentiation.

Genome-Wide Profiling Reveals Alternative Polyadenylation of Innate Immune-Related mRNA in Patients With COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused many deaths worldwide. To date, the mechanism of viral immune escape remains unclear, which is a great obstacle to developing effective clinical treatment. RNA processing mechanisms, including alternative polyadenylation (APA) and alternative splicing (AS), are crucial in the regulation of most human genes in many types of infectious diseases. Because the role of APA and AS in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown, we performed de novo identification of dynamic APA sites using a public dataset of human peripheral blood mononuclear cell (PBMC) RNA-Seq data in COVID-19 patients. We found that genes with APA were enriched in innate immunity -related gene ontology categories such as neutrophil activation, regulation of the MAPK cascade and cytokine production, response to interferon-gamma and the innate immune response. We also reported genome-wide AS events and enriched viral transcription-related categories upon SARS-CoV-2 infection. Interestingly, we found that APA events may give better predictions than AS in COVID-19 patients, suggesting that APA could act as a potential therapeutic target and novel biomarker in those patients. Our study is the first to annotate genes with APA and AS in COVID-19 patients and highlights the roles of APA variation in SARS-CoV-2 infection.

2007 – DIFFERENTIAL ALTERNATIVE POLYADENYLATION LANDSCAPES MEDIATE HEMATOPOIETIC STEM CELL ACTIVATION AND REGULATE GLUTAMINE METABOLISM

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3'-untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies in vivo remains elusive. Using extensive in vitro and in vivo analysis approaches we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Further, we performed low input 3'-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors determining the genome-wide APA landscape (APAome). This revealed transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation (Sommerkamp et al., Cell Stem Cell, 2020). Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching. This process is mediated by Nudt21 and is required for the proper stress response of HSCs. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Inhibition of this metabolic adjustment leads to impaired HSC function and a partial block in differentiation. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal and commitment.

Extensive Involvement of Alternative Polyadenylation in Single-Nucleus Neurons.

Cleavage and polyadenylation are essential processes that can impact many aspects of mRNA fate. Most eukaryotic genes have alternative polyadenylation (APA) events. While the heterogeneity of mRNA polyadenylation isoform choice has been studied in specific tissues, less attention has been paid to the neuronal heterogeneity of APA selection at single-nucleus resolution. APA is highly controlled during development and neuronal activation, however, to what extent APA events vary in a specific neuronal cell population and the regulatory mechanisms are still unclear. In this paper, we investigated dynamic APA usage in different cell types using snRNA-seq data of 1424 human brain cells generated by single-cell 3′ RNA sequencing. We found that distal APA sites are not only favored by global neuronal cells, but that their usage also varies between the principal types of neuronal cell populations (excitatory neurons and inhibitory neurons). A motif analysis and a gene functional analysis indicated the enrichment of RNA-binding protein (RBP) binding sites and neuronal functions for the set of genes with neuron-enhanced distal PAS usage. Our results revealed the extensive involvement of APA regulation in neuronal populations at the single-nucleus level, providing new insights into roles for APA in specific neuronal cell populations, as well as utility in future functional studies.

PolyA-miner: accurate assessment of differential alternative poly-adenylation from 3'Seq data using vector projections and non-negative matrix factorization.

Almost 70% of human genes undergo alternative polyadenylation (APA) and generate mRNA transcripts with varying lengths, typically of the 3′ untranslated regions (UTR). APA plays an important role in development and cellular differentiation, and its dysregulation can cause neuropsychiatric diseases and increase cancer severity. Increasing awareness of APA’s role in human health and disease has propelled the development of several 3′ sequencing (3′Seq) techniques that allow for precise identification of APA sites. However, despite the recent data explosion, there are no robust computational tools that are precisely designed to analyze 3′Seq data. Analytical approaches that have been used to analyze these data predominantly use proximal to distal usage. With about 50% of human genes having more than two APA isoforms, current methods fail to capture the entirety of APA changes and do not account for non-proximal to non-distal changes. Addressing these key challenges, this study demonstrates PolyA-miner, an algorithm to accurately detect and assess differential alternative polyadenylation specifically from 3′Seq data. Genes are abstracted as APA matrices, and differential APA usage is inferred using iterative consensus non-negative matrix factorization (NMF) based clustering. PolyA-miner accounts for all non-proximal to non-distal APA switches using vector projections and reflects precise gene-level 3′UTR changes. It can also effectively identify novel APA sites that are otherwise undetected when using reference-based approaches. Evaluation on multiple datasets—first-generation MicroArray Quality Control (MAQC) brain and Universal Human Reference (UHR) PolyA-seq data, recent glioblastoma cell line NUDT21 knockdown Poly(A)-ClickSeq (PAC-seq) data, and our own mouse hippocampal and human stem cell-derived neuron PAC-seq data—strongly supports the value and protocol-independent applicability of PolyA-miner. Strikingly, in the glioblastoma cell line data, PolyA-miner identified more than twice the number of genes with APA changes than initially reported. With the emerging importance of APA in human development and disease, PolyA-miner can significantly improve data analysis and help decode the underlying APA dynamics.

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

ALTERNATIVE POLYADENYLATION REGULATES HEMATOPOIETIC STEM CELL METABOLISM

Alternative Polyadenylation (APA) is emerging as an important regulatory mechanism controlling 3’-UTRs and thereby RNA and protein isoform expression. However, the relevance of APA in stem cell hierarchies in vivo remains unexplored. Here, we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Furthermore, we performed low input 3’-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors. The 3’-UTRs of HSCs undergo shortening upon transition from quiescence to proliferation during homeostasis and in response to inflammation as well as during HSC differentiation. Strikingly, we observe APA-mediated metabolic reprogramming upon HSC activation leading to an increase in glutaminolysis. This increase is mediated by APA regulators and required for proper HSC function. Inhibition of this metabolic adjustment by targeting APA-regulators leads to impaired HSC function and a partial block in differentiation. Our study establishes APA and associated adaptations of the glutamine metabolism as novel layers of regulation that orchestrate HSC behavior and commitment in the hematopoietic cascade.