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.

Life stage-specific poly(A) site selection regulated by Trypanosoma brucei DRBD18.

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.

The role of alternative polyadenylation in breast cancer.

Breast cancer (BC), as a highly prevalent malignant tumor worldwide, is still unclear in its pathogenesis and has poor therapeutic outcomes. Alternative polyadenylation (APA) is a post-transcriptional regulatory mechanism widely found in eukaryotes. Precursor mRNA (pre-mRNA) undergoes the APA process to generate multiple mRNA isoforms with different coding regions or 3'UTRs, thereby greatly increasing the diversity and complexity of the eukaryotic transcriptome and proteome. Studies have shown that APA is involved in the progression of various diseases, including cancer, and plays a crucial role. Therefore, clarifying the biological mechanisms of APA and its regulators in breast cancer will help to comprehensively understand the pathogenesis of breast cancer and provide new ideas for its prevention and treatment.

Multiplexed single-cell characterization of alternative polyadenylation regulators.

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.

Proximal termination generates a transcriptional state that determines the rate of establishment of Polycomb silencing.

The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.

2'-O-methylation at internal sites on mRNA promotes mRNA stability.

2'-O-methylation (Nm) is a prominent RNA modification well known in noncoding RNAs and more recently also found at many mRNA internal sites. However, their function and base-resolution stoichiometry remain underexplored. Here, we investigate the transcriptome-wide effect of internal site Nm on mRNA stability. Combining nanopore sequencing with our developed machine learning method, NanoNm, we identify thousands of Nm sites on mRNAs with a single-base resolution. We observe a positive effect of FBL-mediated Nm modification on mRNA stability and expression level. Elevated FBL expression in cancer cells is associated with increased expression levels for 2'-O-methylated mRNAs of cancer pathways, implying the role of FBL in post-transcriptional regulation. Lastly, we find that FBL-mediated 2'-O-methylation connects to widespread 3' UTR shortening, a mechanism that globally increases RNA stability. Collectively, we demonstrate that FBL-mediated Nm modifications at mRNA internal sites regulate gene expression by enhancing mRNA stability.

Global analysis of key post-transcriptional regulation in early leaf development of Limonium bicolor identifies a long non-coding RNA that promotes salt gland development and salt resistance.

Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide abundant transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Disruption of PABPN1 phase separation by SNRPD2 drives colorectal cancer cell proliferation and migration through promoting alternative polyadenylation of CTNNBIP1.

Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized. Here, with profiling of APA in colorectal cancer tissues and poly(A) signal editing, we firstly identified that the shortened 3' UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration. We found that liquid-liquid phase separation (LLPS) of PABPN1 is reduced albeit with higher expression in cancer, and the reduction of LLPS leads to the shortened 3' UTR of CTNNBIP1 and promotes cell proliferation and migration. Notably, the splicing factor SNRPD2 upregulated in colorectal cancer, can interact with glutamic-proline (EP) domain of PABPN1, and then disrupt LLPS of PABPN1, which attenuates the repression effect of PABPN1 on the proximal poly(A) sites. Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1, suggesting that regulation of APA by interfering LLPS of 3' end processing factor may have the potential as a new way for the treatment of cancer.

CPSF3 regulates alternative polyadenylation of CNIH2 to promote esophageal squamous cell carcinoma progression.

Alternative polyadenylation (APA), an important post-transcriptional regulatory mechanism, is aberrantly activated in cancer，but how APA functions in tumorigenesis remains elusive. We analyzed APA events in RNA-seq data in TCGA and reported 3'UTR alterations associated with esophageal squamous cell carcinoma (ESCC) patient prognosis and gene expression changes involving loss of tumor-suppressive miRNA binding sites. Moreover, we investigated the expression and function of cleavage and polyadenylation specific factor 3 (CPSF3), a key APA regulator in ESCC. By immunohistochemistry and qRT-PCR, we found that CPSF3 was highly expressed in ESCC tissues and associated with poor patient prognosis. Overexpression of CPSF3 enhanced, while knockdown of CPSF3 inhibited ESCC cell proliferation and migration in vitro and in vivo, as determined by colony formation, transwell assays and animal experiments. Iso-Seq and RNA-seq data analysis indicated that knockdown of CPSF3 favored use of the distal poly (A) site in the 3'UTR of Cornichon family AMPA receptor auxiliary protein 2 (CNIH2), resulting in a long-3'UTR CNIH2 isoform that produced less CNIH2 protein due to miR-125a-5p targeting and downregulating CNIH2 mRNA through a miR-125a-5p binding site in the long CNIH2 mRNA 3'UTR. Moreover, CPSF3-induced ESCC tumorigenicity was mediated by CNIH2. Taken together, CPSF3 promotes ESCC progression by upregulating CNIH2 expression through loss of miR-125a-5p-mediated CNIH2 repression through alternative splicing and polyadenylation of the CNIH2 mRNA 3'UTR.

Alternative polyadenylation quantitative trait loci contribute to acute myeloid leukemia risk genes regulation.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a high relapse rate and progressive drug resistance. Alternative polyadenylation (APA) contributes to post-transcriptional dysregulation, but little is known about the association between APA and AML. The APA quantitative trait locus (apaQTL) is a powerful method to investigate the relationship between APA and single nucleotide polymorphisms (SNPs). We quantified APA usage in 195 Chinese AML patients and identified 4922 cis-apaQTLs related to 1875 genes, most of which were newly reported. Cis-apaQTLs may modulate the APA selection of 115 genes through poly(A) signals. Colocalization analysis revealed that cis-apaQTLs colocalized with cis-eQTLs may regulate gene expression by affecting miRNA binding sites or RNA secondary structures. We discovered 207 cis-apaQTLs related to AML risk by comparing genotype frequency with the East Asian healthy controls from the 1000 Genomes Project. Genes with cis-apaQTLs were associated with hematological phenotypes and tumor incidence according to the PHARMGKB and MGI databases. Collectively, we profiled an atlas of cis-apaQTLs in Asian AML patients and found their association with APA selection, gene expression, AML risk, and complex traits. Cis-apaQTLs may provide insights into the regulatory mechanisms related to APA in AML occurrence, progression, and prognosis.

Alternative Polyadenylation Regulatory Factors Signature for Survival Prediction in Kidney Renal Cell Carcinoma.

Background: Alternative polyadenylation (APA) plays a vital regulatory role in various diseases. It is widely accepted that APA is regulated by APA regulatory factors. Objective: Whether APA regulatory factors affect the prognosis of renal cell carcinoma remains unclear, and this is the main topic of this study. Methods: We downloaded the transcriptome and clinical data from The Cancer Genome Atlas (TCGA) database. We used the Lasso regression system to construct an APA model for analyzing the relationship between common APA regulatory factors and renal cell carcinoma. We also validated our APA model using independent GEO datasets (GSE29609, GSE76207). Results: It was found that the expression levels of 5 APA regulatory factors (CPSF1, CPSF2, CSTF2, PABPC1, and PABPC4) were significantly associated with tumor gene mutation burden (TMB) score in renal clear cell carcinoma, and the risk score constructed using the expression level of 5 key APA regulatory factors could be used to predict the outcome of renal clear cell carcinoma. The TMB score is associated with the remodeling of the immune microenvironment. Conclusions: By identifying key APA regulatory factors in renal cell carcinoma and constructing risk scores for key APA regulatory factors, we showed that key APA regulators affect prognosis of renal clear cell carcinoma patients. In addition, the risk score level is associated with TMB, indicating that APA may affect the efficacy of immunotherapy through immune microenvironment-related genes. This helps us better understand the mRNA processing mechanism of renal clear cell carcinoma.

Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera.

BACKGROUND: Alternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio's SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera. RESULTS: We identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA's role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis. CONCLUSIONS: This study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus.

In gestational diabetes mellitus (GDM), adipose tissue undergoes metabolic disturbances and chronic low-grade inflammation. Alternative polyadenylation (APA) is a post-transcriptional modification mechanism that generates mRNA with variable lengths of 3' untranslated regions (3'UTR), and it is associated with inflammation and metabolism. However, the role of APA in GDM adipose tissue has not been well characterized. In this study, we conducted transcriptomic and proteomic sequencing on subcutaneous and omental adipose tissues from both control and GDM patients. Using Dapars, a novel APA quantitative algorithm, we delineated the APA landscape of adipose tissue, revealing significant 3'UTR elongation of mRNAs in the GDM group. Omental adipose tissue exhibited a significant correlation between elongated 3'UTRs and reduced translation levels of genes related to metabolism and inflammation. Validation experiments in THP-1 derived macrophages (TDMs) demonstrated the impact of APA on translation levels by overexpressing long and short 3'UTR isoforms of a representative gene LRRC25. Additionally, LRRC25 was validated to suppress proinflammatory polarization in TDMs. Further exploration revealed two underexpressed APA trans-acting factors, CSTF3 and PPP1CB, in GDM omental adipose tissue. In conclusion, this study provides preliminary insights into the APA landscape of GDM adipose tissue. Reduced APA regulation in GDM omental adipose tissue may contribute to metabolic disorders and inflammation by downregulating gene translation levels. These findings advance our understanding of the molecular mechanisms underlying GDM-associated adipose tissue changes.

Colocalization analysis of 3' UTR alternative polyadenylation quantitative trait loci reveals novel mechanisms underlying associations with lung function.

While many disease-associated single nucleotide polymorphisms (SNPs) are expression quantitative trait loci (eQTLs), a large proportion of genome-wide association study (GWAS) variants are of unknown function. Alternative polyadenylation (APA) plays an important role in posttranscriptional regulation by allowing genes to shorten or extend 3' untranslated regions (UTRs). We hypothesized that genetic variants that affect APA in lung tissue may lend insight into the function of respiratory associated GWAS loci. We generated alternative polyadenylation (apa) QTLs using RNA sequencing and whole genome sequencing on 1241 subjects from the Lung Tissue Research Consortium (LTRC) as part of the NHLBI TOPMed project. We identified 56 179 APA sites corresponding to 13 582 unique genes after filtering out APA sites with low usage. We found that a total of 8831 APA sites were associated with at least one SNP with q-value < 0.05. The genomic distribution of lead APA SNPs indicated that the majority are intronic variants (33%), followed by downstream gene variants (26%), 3' UTR variants (17%), and upstream gene variants (within 1 kb region upstream of transcriptional start site, 10%). APA sites in 193 genes colocalized with GWAS data for at least one phenotype. Genes containing the top APA sites associated with GWAS variants include membrane associated ring-CH-type finger 2 (MARCHF2), nectin cell adhesion molecule 2 (NECTIN2), and butyrophilin subfamily 3 member A2 (BTN3A2). Overall, these findings suggest that APA may be an important mechanism for genetic variants in lung function and chronic obstructive pulmonary disease (COPD).

Deciphering Estrus Expression in Gilts: The Role of Alternative Polyadenylation and LincRNAs in Reproductive Transcriptomics.

The fertility rate and litter size of female pigs are critically affected by the expression of estrus. The objective of this study was to elucidate the regulatory mechanisms of estrus expression by analyzing the differential expression of genes and long intergenic non-coding RNAs (lincRNA), as well as the utilization of alternative polyadenylation (APA) sites, in the vulva and vagina during the estrus and diestrus stages of Large White and indigenous Chinese Mi gilts. Our study revealed that the number of differentially expressed genes (DEG) in the vulva was less than that in the vagina, and the DEGs in the vulva were enriched in pathways such as "neural" pathways and steroid hormone responses, including the "Calcium signaling pathway" and "Oxytocin signaling pathway". The DEGs in the vagina were enriched in the "Metabolic pathways" and "VEGF signaling pathway". Furthermore, 27 and 21 differentially expressed lincRNAs (DEL), whose target genes were enriched in the "Endocrine resistance" pathway, were identified in the vulva and vagina, respectively. Additionally, we observed that 63 and 618 transcripts of the 3'-untranslated region (3'-UTR) were lengthened during estrus in the vulva and vagina, respectively. Interestingly, the genes undergoing APA events in the vulva exhibited species-specific enrichment in neural or steroid-related pathways, whereas those in the vagina were enriched in apoptosis or autophagy-related pathways. Further bioinformatic analysis of these lengthened 3'-UTRs revealed the presence of multiple miRNAs binding sites and cytoplasmic polyadenylation element (CPE) regulatory aspects. In particular, we identified more than 10 CPEs in the validated lengthened 3'-UTRs of the NFIX, PCNX4, CEP162 and ABHD2 genes using RT-qPCR. These findings demonstrated the involvement of APA and lincRNAs in the regulation of estrus expression in female pigs, providing new insights into the molecular mechanisms underlying estrus expression in pigs.

The Transcriptional Landscape of Immune-Response 3'-UTR Alternative Polyadenylation in Melanoma.

The prognosis of patients with malignant melanoma has been improved in recent decades due to advancements in immunotherapy. However, a considerable proportion of patients are refractory to treatment, particularly at advanced stages. This underscores the necessity of developing a new strategy to improve it. Alternative polyadenylation (APA), as a marker of crucial posttranscriptional regulation, has emerged as a major new type of epigenetic marker involved in tumorigenesis. However, the potential roles of APA in shaping the tumor microenvironment (TME) are largely unexplored. Herein, we collected two cohorts comprising melanoma patients who received immune checkpoint inhibitor (ICI) immunotherapy to quantify transcriptome-wide discrepancies in APA. We observed a global change in 3'-UTRs between responders and non-responders, which might involve DNA damage response, angiogenesis, PI3K-AKT signaling pathways, etc. Ten putative master APA regulatory factors for those APA events were detected via a network analysis. Notably, we established an immune response-related APA scoring system (IRAPAss), which exhibited a great performance of predicting immunotherapy response in multiple cohorts. Furthermore, we examined the correlation of APA with TME at the single-cell level using four single-cell immune profiles of tumor-infiltrating lymphocytes (TILs), which revealed an overall discrepancy in 3'-UTR length across diverse T cell populations, probably contributing to immunoregulation in melanoma. In conclusion, our study provides a transcriptional landscape of APA implicated in immunoregulation, which might lay the foundation for developing a new strategy for improving immunotherapy response for melanoma patients by targeting APA.

ASAPA: a bioinformatic pipeline based on Iso-Seq that identifies the links among alternative splicing, alternative transcription initiation and alternative polyadenylation.

BACKGROUND: Although the events associated with alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI) can be identified by many approaches based on isoform sequencing (Iso-Seq), these analyses are generally independent of each other and the links between these events are still rarely mentioned. However, an interdependency analysis can be achieved because the transcriptional start site, splice sites and polyA site could be simultaneously included in a long, full-length read from Iso-Seq. RESULTS: We create ASAPA pipeline that enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data. We tested this pipeline using Arabidopsis data and found some interesting results: some adjacent introns tend to be simultaneously spliced or retained; coupling between AS and ATI or APA is limited to the initial or terminal intron; and ATI and APA are potentially linked in some special cases. CONCLUSION: Our pipeline enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data, which is conducive to a better understanding of transcription landscape generation.