Polyadenylation Site Usage Research Articles

A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage.

Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.

Isoform balance of the long noncoding RNA NEAT1 is regulated by the RNA-binding protein QKI, governs the glioma transcriptome, and impacts cell migration

The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in a variety of human cancers. Two overlapping NEAT1 isoforms, NEAT1_1 and NEAT1_2, are produced through mutually exclusive alternative 3' end formation. Previous studies extensively investigated NEAT1 dysregulation in tumors, but often failed to achieve distinct quantification of the two NEAT1 isoforms. Moreover, molecular mechanisms governing the biogenesis of NEAT1 isoforms and the functional impacts of their dysregulation in tumorigenesis remain poorly understood. In this study, we employed an isoform-specific quantification assay and found differential dysregulation of NEAT1 isoforms in patient-derived glioblastoma multiforme cells. We further showed usage of the NEAT1 proximal polyadenylation site (PAS) is a critical mechanism that controls glioma NEAT1 isoform production. CRISPR-Cas9-mediated PAS deletion reduced NEAT1_1 and reciprocally increased NEAT1_2, which enhanced nuclear paraspeckle formation in human glioma cells. Moreover, the utilization of the NEAT1 PAS is facilitated by the RNA-binding protein quaking (QKI), which binds to the proximal QKI recognition elements. Functionally, we identified transcriptomic changes and altered biological pathways caused by NEAT1 isoform imbalance in glioma cells, including the pathway for the regulation of cell migration. Finally, we demonstrated the forced increase of NEAT1_2 upon NEAT1 PAS deletion is responsible for driving glioma cell migration and promoting the expression of genes implicated in the regulation of cell migration. Together, our studies uncovered a novel mechanism that regulates NEAT1 isoforms and their functional impacts on the glioma transcriptome, which affects pathological pathways of glioma, represented by migration.

Improved characterization of single-cell RNA-seq libraries with paired-end avidity sequencing.

Prevailing poly(dT)-primed 3' single-cell RNA-seq protocols generate barcoded cDNA fragments containing the reverse transcriptase priming site, which is expected to be the poly(A) tail or a genomic adenine homopolymer. Direct sequencing across this priming site was historically difficult because of DNA sequencing errors induced by the homopolymeric primer at the 'barcode' end. Here, we evaluate the capability of "avidity base chemistry" DNA sequencing from Element Biosciences to sequence through this homopolymer accurately, and the impact of the additional cDNA sequence on read alignment and precise quantification of polyadenylation site usage. We find that the Element Aviti instrument sequences through the thymine homopolymer into the subsequent cDNA sequence without detectable loss of accuracy. The resulting paired-end alignments enable direct and independent assignment of reads to polyadenylation sites, which bypasses complexities and limitations of conventional approaches but does not consistently improve read mapping rates compared to single-end alignment. We also characterize low-level artifacts and arrive at an adjusted adapter trimming and alignment workflow that significantly improves the alignment of sequence data from Element and Illumina, particularly in the context of extended read lengths. Our analyses confirm that Element avidity sequencing is an effective alternative to Illumina sequencing for standard single-cell RNA-seq, particularly for polyadenylation site analyses but do not rule out the potential for similar performance from other emerging platforms.

A Developmental Mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage.

Alternative Cleavage and Polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3'UTRs from the same genetic locus, potentially impacting mRNA translation, localization and stability. Developmentally regulated APA can thus make major contributions to cell-type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, approximately 500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of Cleavage Factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knock down of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell-type-specific APA at selected genes.

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.

CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

Platinum-based chemotherapy is the standard postoperative adjuvant treatment for ovarian cancer (OC). Despite the initial response to chemotherapy, 85% of advanced OC patients will have recurrent disease. Relapsed disease and platinum resistance are the major causes of death in OC patients. In this study, we compared the global regulation of alternative polyadenylation (APA) in platinum-resistant and platinum-sensitive tissues of OC patients by analyzing a set of single-cell RNA sequencing (scRNA-seq) data from public databases and found that platinum-resistant patients exhibited global 3’ untranslated region (UTR) shortening due to the different usage of polyadenylation sites (PASs). The APA regulator CSTF3 was the most significantly upregulated gene in epithelial cells of platinum-resistant OC. CSTF3 knockdown increased the sensitivity of OC cells to platinum. The lncRNA NEAT1 has two isoforms, short (NEAT1_1) and long (NEAT1_2) transcript, because of the APA processing in 3’UTR. We found that CSTF3 knockdown reduced the usage of NEAT1 proximal PAS to lengthen the transcript and facilitate the expression of NEAT1_2. Downregulation of the expression of NEAT1 (NEAT1_1/_2), but not only NEAT1_2, also increased the sensitivity of OC cells to platinum. Overexpressed NEAT1_1 reversed the platinum resistance of OC cells after knocking down CSTF3 expression. Furthermore, downregulated expression of CSTF3 and NEAT1_1, rather than NEAT1_2, was positively correlated with inactivation of the PI3K/AKT/mTOR pathway in OC cells. Together, our findings revealed a novel mechanism of APA regulation in platinum-resistant OC. CSTF3 directly bound downstream of the NEAT1 proximal PAS to generate the short isoform NEAT1_1 and was conducive to platinum resistance, which provides a potential biomarker and therapeutic strategy for platinum-resistant OC patients.

Uncovering the dynamics and consequences of RNA isoform changes during neuronal differentiation

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.

Abstract 3301: Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy

Abstract Gene dysregulation is a hallmark of cancer, arising from genetic alterations that invariably lead to dysregulated transcriptional programs. This dysregulation renders cancer cells highly dependent on certain gene expression regulators, leading to a potential for novel therapeutic strategies. Consequently, ongoing research aims to develop small molecules targeting RNA transcriptional processes. Cyclin-dependent kinase 12/13 (CDK12/13) is a member of the cyclin-dependent kinase family of serine/threonine protein kinases. CDK12/13 regulates the RNA transcription elongation by phosphorylating RNA polymerase II and promotes the elongation of transcripts, specifically those involved in DNA damage responses, such as BRCA1/2. The inhibition of CDK12/13 is expected to exert a synergistic effect when combined with PARP inhibitors and chemotherapeutic reagents. Therefore, we developed a novel small molecule inhibitor, CTX-439 (former name: CRD-1835439). Additionally, we demonstrated that CTX-439 exhibits a preclinical efficacy in vitro and in vivo [Cancer Res. (2022) 82 (12_Supplement): 5485]. To identify potential strategies for a clinical trial of CTX-439, in the current study, we conducted translational research to elucidate (1) pharmacodynamics (PD) markers to monitor the suppression of CDK12/13 in humans, (2) patient selection biomarkers to determine sensitivity to CTX-439, and (3) combination strategies with other key medications. Firstly, to identify PD markers of CTX-439, we performed a comprehensive analysis employing RNA-seq, Pol II-ChIP-seq, and Poly(A)-seq. We confirmed that CTX-439 induced the usage of intronic polyadenylation sites by inhibiting CDK12, resulting in the production of shortened mRNA. Of these transcripts, PCF11 emerged as a potential PD marker for clinical trials, which was confirmed using qPCR analysis of human blood. Secondly, to clarify the indication of CTX-439, we performed an in vitro survey involving 400 cell lines and 100 PDX-derived organoids (PDXOs) to identify biomarkers displaying sensitivity and insensitivity to CTX-439. Furthermore, a significant anti-tumor effect of CTX-439 was revealed in PDX models that showed sensitivity in the survey. CTX-439 exhibited a synergistic effect in combination with PARP inhibitors both in vitro and in vivo, highlighting its potential for use in combination strategies. Additionally, CTX-439 showed an anti-tumor effect when combined with other chemotherapy and molecular-targeted drugs. Collectively, the results from these translational research studies provide a foundation for developing a clinical trial by clearly defining PD monitoring, patient stratification biomarkers, and combination strategies. These findings highlight the therapeutic potential of CTX-439 as a single-agent and in combination with inhibitors, such as PARP, for the treatment of cancers characterized by transcriptional addiction. Citation Format: Hiroko Yamakawa, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa, Kosuke Yusa, Daisuke Morishita. Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3301.

Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer

Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 were not previously reported. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-β/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine.

The association of the RSC remodeler complex with chromatin is influenced by the prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

LINC00921 Diminishes Lung Cancer Radiosensitivity by Bestabilizing NUDT21 and Driving Aberrant MED23 Alternative Polyadenylation

Alternative polyadenylation (APA) plays a major role in controlling transcriptome diversity and therapeutic resistance of cancers. However, long-noncoding RNAs (lncRNAs) involved in pathological APA remain poorly defined. Here, we functionally identified a MED13L/P300-induced oncogenic lncRNA, LINC00921, diminished lung cancer radiosensitivity by destabilizing NUDT21 and driving aberrant MED23 alternative polyadenylation. ChIP-seq screening, RNA-seq and real-time PCR were used to identified LINC00921 in NSCLC. We performed RNA pulldown, RIP-qPCR, western blotting and Co-immunoprecipitation to investigate the function of LINC00921, which induced destabilization of NUDT21 and promoted 3' UTR shortening of MED23 via APA. Through H3K27ac ChIP-seq screening, we functionally characterize LINC00921, a MED13L/P300-induced oncogenic lncRNA, required for global regulation of APA in non-small-cell lung cancer (NSCLC). LINC00921 shows significant potential for reducing radiosensitivity of NSCLC and high LINC00921 levels were associated with poor prognosis for NSCLC patients treated with radiotherapy. Mechanistically, LINC00921 directly interacts withNUDT21 via binding to its RNA-binding motif-2. LINC00921 controls NUDT21 stability via facilitating binding of NUDT21 with its newly identified E3 ligase TRIP12. Intriguingly, 3'UTR APA profiles reveal that LINC00921-induced destabilization of NUDT21 decreases the percentage of distal polyadenylation sites (PAS) usage index, resulting in the 3' UTR shortening of MED23 mRNA, which, in turn, leads to elevated MED23 protein levels in cancer cells. MED23 further increases nuclear translocation of β-catenin, and, thereby, activates expression of multiple β-Catenin/TCF/LEF-regulated core oncogenes (c-Myc, CCND1, and BMP4). Taken together, our data revealed a novel model that integrates a lncRNA into regulation of malignant APA, radiotherapy resistance and NSCLC progression. These findings highlight the importance of functionally annotating lncRNAs controlling APA and unlock the clinical potential of novel therapeutics for advanced NSCLC.

Divergent molecular events underlying initial T-cell commitment in human prenatal and postnatal thymus.

Intrathymic T-cell development is a coordinated process accompanied by dynamic changes in gene expression. Although the transcriptome characteristics of developing T cells in both human fetal and postnatal thymus at single-cell resolution have been revealed recently, the differences between human prenatal and postnatal thymocytes regarding the ontogeny and early events of T-cell development still remain obscure. Moreover, the transcriptional heterogeneity and posttranscriptional gene expression regulation such as alternative polyadenylation at different stages are also unknown. In this study, we performed integrative single-cell analyses of thymocytes at distinct developmental stages. The subsets of prenatal CD4-CD8- double-negative (DN) cells, the most immature thymocytes responsible for T-cell lineage commitment, were characterized. By comprehensively comparing prenatal and postnatal DN cells, we revealed significant differences in some key gene expressions. Specifically, prenatal DN subpopulations exhibited distinct biological processes and markedly activated several metabolic programs that may be coordinated to meet the required bioenergetic demands. Although showing similar gene expression patterns along the developmental path, prenatal and postnatal thymocytes were remarkably varied regarding the expression dynamics of some pivotal genes for cell cycle, metabolism, signaling pathway, thymus homing, and T-cell commitment. Finally, we quantified the transcriptome-wide changes in alternative polyadenylation across T-cell development and found diverse preferences of polyadenylation site usage in divergent populations along the T-cell commitment trajectory. In summary, our results revealed transcriptional heterogeneity and a dynamic landscape of alternative polyadenylation during T-cell development in both human prenatal and postnatal thymus, providing a comprehensive resource for understanding T lymphopoiesis in human thymus.

The promoter as a trip navigator: Guiding alternative polyadenylation site destinations

Alfonso-Gonzalez etal.1 present an innovative combination of long-read-sequencing approaches that reveal coupling of alternative transcription start sites and alternative polyadenylation site usage on a global level.

Abstract B060: Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities

Abstract Background: CDK12 expression is lost in ~5% of metastatic castration-resistant prostate cancer (mCRPC) and associates with aggressive disease. Previous literature, based on acute CDK12-loss models, proposes a mechanism where CDK12 loss leads to a homologous recombination deficiency (HRD) phenotype via premature intronic polyadenylation of classic HR pathway genes, including BRCA1 and BRCA2. Cyclin K is an essential gene and functions in a heterodimer with CDK12 or CDK13. We sought to test if CDK12 loss: (1) confers HRD and a corresponding sensitivity to platinum and poly-ADP ribose polymerase (PARP) inhibitors and/or (2) confers synthetic lethality to other cellular targets and pathways. Methods: To investigate the role of CDK12 loss we used a combination of acute (Tet-inducible shRNA, CDK12/13 inhibitors) and adapted (naturally mutant LuCaP 189.4, CRISPR KO clones) models. DNA repair gene expression and polyadenylation site usage was measured by RNA-seq, qPCR, and western blot while HR function was measured by immunofluorescence for RAD51 foci following irradiation. Stable CDK12-negative (CDK12-neg) cell lines were treated with sgRNAs, PARP inhibitors, carboplatin, and CDK inhibitors to test for potential targeted vulnerabilities. Results: Acute CDK12 inhibition (6h, SR4835) confirmed the reported mechanism of 3’ transcript loss, but the effects were minimal in long term shCDK12 or CRISRP KO lines as measured by RNA-seq, qPCR, and western blot. CDK12-neg lines showed slight decreases in ATR and ATM protein but minimal changes in BRCA1 or BRCA2 and no decrease in RAD51 foci formation. Furthermore, these lines did not show increased sensitivity to carboplatin and only partial sensitivity to PARPi. Human tumor data were analyzed and showed that CDK12-neg tumors lack genomic signatures of HRD and do not show significant de-enrichment of long genes. However, CDK12-neg cell lines did show sensitivity to CDK13 KO or pharmacologic inhibition with CDK12/13 inhibitors (SR4835, THZ531, dinaciclib). In vivo treatment of LuCaP xenograft lines showed that 189.4 responded to SR4835, while two CDK12-intact lines (35 and 136) did not. Conclusions: Our data support a model where HRD is primarily a result of acute CDK12 loss, while the downregulation of long DNA repair genes is largely compensated for (via currently unknown mechanisms) in cells that have adapted to CDK12 loss. We suspect that the partial PARPi sensitivity observed in CDK12-neg lines is due to other (non-HRD) DNA repair deficiencies and potential mechanisms are under investigation. CDK12-neg cells exhibited dependency on CDK13, which can be targeted with semi-selective pharmacologic compounds, though our data support the development of specific CDK13 inhibitors to maximize synthetic lethality and minimize off-target effects. Finally, additional work is ongoing with CRISPR and drug screens to identify additional vulnerabilities in adapted CDK12-neg models and provide future clinical targets for this aggressive subset of mCRPC. Citation Format: Sander Frank, Ilsa Coleman, Navonil De Sarkar, Dmytro Rudoy, Valeri Vasioukhin, Pete Nelson. Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B060.

Abstract 277: NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform

Abstract Background: Metabolic dysregulation, a hallmark of cancer, allows tumor cells to sustain high rates of growth and proliferation in unfavorable conditions, including hypoxia. Many cancers, including clear cell renal cell carcinoma (ccRCC), demonstrate the Warburg effect with high rates of glycolysis and some are dependent on glutamine. These cancers express high levels of glutaminase (GLS), the enzyme responsible for conversion of glutamine to glutamate. GLS exists as two distinct isoforms, GAC and KGA, which are generated by alternative polyadenylation. The presence of GAC favors more metabolically active cell growth, and the GAC:KGA ratio negatively correlates with survival in ccRCC. Selectively inhibiting the GAC isoform may be a target for therapy. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), has shown encouraging efficacy signals in several solid tumor types in a Phase ½ clinical study (NuTide:701 NCT03829254). It generates 3’-dATP which profoundly affects transcription by altering polyadenylation and has been shown to alter expression of mitochondrial electron transport chain genes. The aim of this study was to investigate the impact of NUC-7738 on GLS alternative polyadenylation and the ratio of GAC:KGA isoforms. Material and Methods: ccRCC (Caki-1 [VHL mutant];786-O [wildtype]) and pancreatic (PANC1; MIAPACA) cancer cell lines were treated with NUC-7738 at IC50 doses in hypoxic and normoxic conditions. NUC-7738 and 3’-dATP were measured by LC-MS. mRNA levels of glutaminase isoforms were determined by RT-qPCR using primers targeting exon 14-15 junction for GAC and exon 16-18 junction for KGA. GAC and KGA protein expression determined by JESS Western analysis. Results: NUC-7738 was converted into 3’-dATP within 6 hours of treatment with an average concentration of 8 pmol/106 cells, which was maintained for ~24 hours and decreased by 50% by 48 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. In ccRCC cells (VHL wildtype and mutant), NUC-7738 reduced transcript expression of GAC isoform, with no change in KGA, indicating a change in polyadenylation site usage. This was reflected in altered protein levels, with decreased GAC in ccRCC and pancreatic cell lines in hypoxic and normoxic conditions after NUC-7738 treatment. Conclusion: NUC-7738 generates sustained levels of 3’-dATP in cells, which is associated with alternative polyadenylation site usage changes. In a variety of cell types, mRNA and protein levels of the more metabolically active GAC isoform were reduced, with an increase in the relative amount of KGA isoform. Increased glutamate production and GAC:KGA ratio are key for survival of some tumors. As alternative polyadenylation is implicated in the control of expression of many genes in cancer, the ability to influence it may lead to new strategies for developing anti-cancer treatments interfering with cellular metabolism. Citation Format: Mustafa Elshani, Ying Zhang, Tia Hawkins, Mary Kudsy, In Hwa Um, Greice Zickuhr, Alison L. Dickson, David J. Harrison. NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 277.

Accurate transcriptome-wide identification and quantification of alternative polyadenylation from RNA-seq data with APAIQ.

Alternative polyadenylation (APA) enables a gene to generate multiple transcripts with different 3' ends, which is dynamic across different cell types or conditions. Many computational methods have been developed to characterize sample-specific APA using the corresponding RNA-seq data, but suffered from high error rate on both polyadenylation site (PAS) identification and quantification of PAS usage (PAU), and bias toward 3' untranslated regions. Here we developed a tool for APA identification and quantification (APAIQ) from RNA-seq data, which can accurately identify PAS and quantify PAU in a transcriptome-wide manner. Using 3' end-seq data as the benchmark, we showed that APAIQ outperforms current methods on PAS identification and PAU quantification, including DaPars2, Aptardi, mountainClimber, SANPolyA, and QAPA. Finally, applying APAIQ on 421 RNA-seq samples from liver cancer patients, we identified >540 tumor-associated APA events and experimentally validated two intronic polyadenylation candidates, demonstrating its capacity to unveil cancer-related APA with a large-scale RNA-seq data set.

Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage.

Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.

CPSF6-mediated XBP1 3’UTR shortening attenuates cisplatin-induced ER stress and elevates chemo-resistance in lung adenocarcinoma

Alternative polyadenylation (APA) is a widespread mechanism generating RNA molecules with alternative 3' ends. Herein, we discovered that TargetScan includes a novel XBP1 transcript with a longer 3' untranslated region (UTR) (XBP1-UL) than that included in NCBI. XBP1-UL exhibited a lowered level in blood samples from lung adenocarcinoma (LUAD) patients and in those after DDP treatment. Consistently, XBP1-UL was reduced in A549 cells compared to normal BEAS-2B cells, as well as in DDP-treated/resistant A549 cells relative to controls. Moreover, due to decreased usage of the distal polyadenylation site (PAS) in 3’UTR, XBP1-UL level was lowered in A549 cells and decreased further in DDP-resistant A549 (A549/DDP) cells. Importantly, use of the distal PAS (dPAS) and XBP1-UL level were gradually reduced in A549 cells under increasing concentrations of DDP, which was attributed to DDP-induced endoplasmic reticulum (ER) stress. Furthermore, XBP1 transcripts with shorter 3’UTR (XBP1-US) were more stable and presented stronger potentiation on DDP resistance. The choice of proximal PAS (pPAS) was attributed to CPSF6 elevation, which was caused by BRCA1-distrupted R-loop accumulation in CPSF6 5’end. DDP-induced nuclear LINC00221 also facilitated CPSF6-induced pPAS choice in the pre-XBP1 3’end. Finally, we found that unlike the unspliced XBP1 protein (XBP1-u), the spliced form XBP1-s retarded p53 degradation to facilitate DNA damage repair of LUAD cells. The current study provides new insights into tumor progression and DDP resistance in LUAD, which may contribute to improved LUAD treatment.

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.

Single Nucleus Transcriptome Data from Alzheimer's Disease Mouse Models Yield New Insight into Pathophysiology.

5XFAD humanized mutant mice and Trem2 knockout (T2KO) mice are two mouse models relevant to the study of Alzheimer's disease (AD)-related pathology. To determine hippocampal transcriptomic and polyadenylation site usage alterations caused by genetic mutations engineered in 5XFAD and T2KO mice. Employing a publicly available single-nucleus RNA sequencing dataset, we used Seurat and Sierra analytic programs to identify differentially expressed genes (DEGs) and differential transcript usage (DTU), respectively, in hippocampal cell types from each of the two mouse models. We analyzed cell type-specific DEGs further using Ingenuity Pathway Analysis (IPA). We identified several DEGs in both neuronal and glial cell subtypes in comparisons of wild type (WT) versus 5XFAD and WT versus T2KO mice, including Ttr, Fth1, Pcsk1n, Malat1, Rpl37, Rtn1, Sepw1, Uba52, Mbp, Arl6ip5, Gm26917, Vwa1, and Pgrmc1. We also observed DTU in common between the two comparisons in neuronal and glial subtypes, specifically in the genes Prnp, Rbm4b, Pnisr, Opcml, Cpne7, Adgrb1, Gabarapl2, Ubb, Ndfip1, Car11, and Stmn4. IPA identified three statistically significant canonical pathways that appeared in multiple cell types and that overlapped between 5XFAD and T2KO comparisons to WT, including 'FXR/RXR Activation', 'LXR/RXR Activation', and 'Acute Phase Response Signaling'. DEG, DTU, and IPA findings, derived from two different mouse models of AD, highlight the importance of energy imbalance and inflammatory processes in specific hippocampal cell types, including subtypes of neurons and glial cells, in the development of AD-related pathology. Additional studies are needed to further characterize these findings.