The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts.

Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

Neuronal Cells Display Distinct Stability Controls of Alternative Polyadenylation mRNA Isoforms, Long Non-Coding RNAs, and Mitochondrial RNAs.

RNA stability plays an important role in gene expression. Here, using 3' end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3'UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3'-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5' splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid 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. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown 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 the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

MAAPER: model-based analysis of alternative polyadenylation using 3' end-linked reads.

Most eukaryotic genes express alternative polyadenylation (APA) isoforms. A growing number of RNA sequencing methods, especially those used for single-cell transcriptome analysis, generate reads close to the polyadenylation site (PAS), termed nearSite reads, hence inherently containing information about APA isoform abundance. Here, we present a probabilistic model-based method named MAAPER to utilize nearSite reads for APA analysis. MAAPER predicts PASs with high accuracy and sensitivity and examines different types of APA events with robust statistics. We show MAAPER's performance with both bulk and single-cell data and its applicability in unpaired or paired experimental designs.

Alternative 3' UTRs play a widespread role in translation-independent mRNA association with the endoplasmic reticulum.

Transcripts encoding membrane and secreted proteins are known to associate with the endoplasmic reticulum (ER) through translation. Here, using cell fractionation, polysome profiling, and 3' end sequencing, we show that transcripts differ substantially in translation-independent ER association (TiERA). Genes in certain functional groups, such as cell signaling, tend to have significantly higher TiERA potentials than others, suggesting the importance of ER association for their mRNA metabolism, such as localized translation. The TiERA potential of a transcript is determined largely by size, sequence content, and RNA structures. Alternative polyadenylation (APA) isoforms can have distinct TiERA potentials because of changes in transcript features. The widespread 3' UTR lengthening in cell differentiation leads to greater transcript association with the ER, especially for genes that are capable of expressing very long 3' UTRs. Our data also indicate that TiERA is in dynamic competition with translation-dependent ER association, suggesting limited space on the ER for mRNA association.

A CRISPR RNA-binding protein screen reveals regulators of RUNX1 isoform generation.

The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and is disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined central role, and mutations promote malignant transformation. More recently, studies have illuminated the importance of posttranscriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and the breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between posttranscriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the posttranscriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a vs RUNX1b/c) that mediate HSC self-renewal vs differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detect changes in RNA processing. We used this reporter in a clustered regularly interspaced short palindromic repeats (CRISPR) screen consisting of single guide RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the posttranscriptional regulation of a key hematopoietic transcription factor and identifies RBPs that may have widespread and important functions in hematopoiesis.

3'READS + RIP defines differential Staufen1 binding to alternative 3'UTR isoforms and reveals structures and sequence motifs influencing binding and polysome association.

Staufen1 (STAU1) is an RNA-binding protein (RBP) that interacts with double-stranded RNA structures and has been implicated in regulating different aspects of mRNA metabolism. Previous studies have indicated that STAU1 interacts extensively with RNA structures in coding regions (CDSs) and 3'-untranslated regions (3'UTRs). In particular, duplex structures formed within 3'UTRs by inverted-repeat Alu elements (IRAlus) interact with STAU1 through its double-stranded RNA-binding domains (dsRBDs). Using 3' region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3'READS + RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3'UTRs. Overall, relative to short 3'UTR counterparts, longer 3'UTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specific IRAlus sequences. Nevertheless, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3'UTRs that may recruit antagonistic RBPs and/or destabilize RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3'UTRs generally enhances polysome association. However, IRAlus generally have little impact on STAU1-mediated polysome association despite having strong interactions with the protein. Taken together, our work reveals complex interactions of STAU1 with its cognate RNA substrates. Our data also shed light on distinct post-transcriptional fates for the widespread APA isoforms in mammalian cells.

Widespread transcript shortening through alternative polyadenylation in secretory cell differentiation.

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.

ALS/FTD-associated protein FUS induces mitochondrial dysfunction by preferentially sequestering respiratory chain complex mRNAs.

Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.

ALYREF links 3'-end processing to nuclear export of non-polyadenylated mRNAs.

The RNA-binding protein ALYREF plays key roles in nuclear export and also 3'-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3'-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-specific component Lsm11. Furthermore, ALYREF, together with other components of the TREX complex, enhances histone mRNA export. Moreover, we show that 3'-end processing promotes ALYREF recruitment and histone mRNA export. Together, our results point to an important role of ALYREF in coordinating 3'-end processing and nuclear export of non-polyadenylated mRNAs.

Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes.

Regulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell.

The mRNA Export Receptor NXF1 Coordinates Transcriptional Dynamics, Alternative Polyadenylation, and mRNA Export.

Alternative polyadenylation (APA) produces mRNA isoforms with different 3' UTR lengths. Previous studies indicated that 3' end processing and mRNA export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to long 3' UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3' UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3' end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3' UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3' end processing, and nuclear export of long 3' UTR transcripts, implicating NXF1 as a nexus of gene regulation.

A compendium of conserved cleavage and polyadenylation events in mammalian genes.

Cleavage and polyadenylation is essential for 3' end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3' UTRs and/or coding sequences. Here, we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on approximately 1.2 billion 3' end sequencing reads from more than 360 human, mouse, and rat samples. We show that ∼80% of mammalian mRNA genes contain at least one conserved PAS, and ∼50% have conserved APA events. PAS conservation generally reduces promiscuous 3' end processing, stabilizing gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with conserved APA events. Whereas tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. In addition, we show enrichment of mRNA destabilizing motifs in alternative 3' UTR sequences, leading to substantial differences in mRNA stability between 3' UTR isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Furthermore, we show that mutations of U-rich motifs around the PAS often accompany APA profile differences between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation through evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic perspective on APA functions.

Cellular stress alters 3'UTR landscape through alternative polyadenylation and isoform-specific degradation.

Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different 3'UTR lengths, production of which is influenced by cellular conditions. Here, we show that arsenic stress elicits global shortening of 3'UTRs through preferential usage of proximal polyadenylation sites during stress and enhanced degradation of long 3'UTR isoforms during recovery. We demonstrate that RNA-binding protein TIA1 preferentially interacts with alternative 3'UTR sequences through U-rich motifs, correlating with stress granule association and mRNA decay of long 3'UTR isoforms. By contrast, genes with shortened 3'UTRs due to stress-induced APA can evade mRNA clearance and maintain transcript abundance post stress. Furthermore, we show that stress causes distinct 3'UTR size changes in proliferating and differentiated cells, highlighting its context-specific impacts on the 3'UTR landscape. Together, our data reveal a global, 3'UTR-based mRNA stability control in stressed cells and indicate that APA can function as an adaptive mechanism to preserve mRNAs in response to stress.

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes.

PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3' region extraction and deep sequencing (3'READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3' ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data.

Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation.

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3' untranslated regions (3'UTRs) and/or coding sequences. Changes in the 3'UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3' end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3'UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3'UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.

Polyadenylation Site-Based Analysis of Transcript Expression by 3'READS.

Deep sequencing of the 3' end region of poly(A)+ RNA identifies the cleavage and polyadenylation site (PAS) and measures transcript abundance. However, mispriming at internal A-rich regions by the oligo-dT oligo in reverse transcription can lead to falsely identified PASs. This problem can be resolved by direct ligation of an adapter to the 3' end of RNA. However, ligation-based methods are often inefficient. Here, we describe 3'READS+, an accurate and sensitive method for deep sequencing of the 3' end of poly(A)+ RNA. Through partial digestion by RNase H of the poly(A) tail bound to a locked nucleic acid (LNA)/DNA hybrid oligo, this method sequences an optimal number of terminal A's, which balances sequencing quality and accurate identification of PAS in A-rich regions. With efficient ligation steps, 3'READS+ is amenable to small amounts of input RNA. 3'READS+ can also be readily used as a cost-effective method for gene expression analysis.

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.

3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

Sequencing of the 3' end of poly(A)(+) RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3' region extraction and deep sequencing (3'READS), to address mispriming issues that often plague 3' end sequencing. Here we report a new version, named 3'READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)(+) RNA and to remove the bulk of the poly(A) tail, 3'READS+ generates RNA fragments with an optimal number of terminal A's that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3'READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (∼50%). 3'READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3' end counting, especially when the amount of input total RNA is limited.