Cleavage And Polyadenylation Research Articles

Light regulates widespread plant alternative polyadenylation through the chloroplast

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

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 42CPA 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.

Cleavage and polyadenylation machinery as a novel targetable vulnerability for human cancer.

The role of alternative polyadenylation of mRNA in sustaining aggressive features of tumors is quite well established, as it is responsible for the 3'UTR shortening of oncogenes and subsequent relief from miRNA-mediated repression observed in cancer cells. However, the information regarding the vulnerability of cancer cells to the inhibition of cleavage and polyadenylation (CPA) machinery is very scattered. Only few recent reports show the antitumor activity of pharmacological inhibitors of CPSF3, one among CPA factors. More in general, the fact that deregulated CPA can be seen as a new hallmark of cancer and as a potential reservoir of novel therapeutic targets has never been formalized. Here, to extend our view on the potential of CPA inhibition (CPAi) approaches as anticancer therapies, we systematically tested the fitness of about one thousand cell lines of different cancer types upon depletion of all known CPA factors by interrogating genome-scale CRISPR and RNAi dependency maps of the DepMap project. Our analysis confirmed core and accessory CPA factors as novel vulnerabilities for human cancer, thus highlighting the potential of CPAi as anticancer therapy. Among all, CPSF1 appeared as a promising actionable candidate for drug development, as it showed low dependency scores pancancer and particularly in highly proliferating cells. In a personalized medicine perspective, the observed differential vulnerability of cancer cell lines to selected CPA factors may be used to build up signatures to predict response of individual human tumors to CPAi approaches.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Elevated pre-mRNA 3′ end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation

Cleavage and polyadenylation (CPA) is responsible for 3′ end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.

Known sequence features explain half of all human gene ends.

Cleavage and polyadenylation (CPA) sites define eukaryotic gene ends. CPA sites are associated with five key sequence recognition elements: the upstream UGUA, the polyadenylation signal (PAS), and U-rich sequences; the CA/UA dinucleotide where cleavage occurs; and GU-rich downstream elements (DSEs). Currently, it is not clear whether these sequences are sufficient to delineate CPA sites. Additionally, numerous other sequences and factors have been described, often in the context of promoting alternative CPA sites and preventing cryptic CPA site usage. Here, we dissect the contributions of individual sequence features to CPA using standard discriminative models. We show that models comprised only of the five primary CPA sequence features give highest probability scores to constitutive CPA sites at the ends of coding genes, relative to the entire pre-mRNA sequence, for 59% of all human genes. U1-hybridizing sequences provide a small boost in performance. The addition of all known RBP RNA binding motifs to the model increases this figure to only 61%, suggesting that additional factors beyond the core CPA machinery have a minimal role in delineating real from cryptic sites. To our knowledge, this high effectiveness of established features to predict human gene ends has not previously been documented.

Sequential Polyadenylation to Enable Alternative mRNA 3' End Formation.

In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual transcripts. Many transcripts are alternatively polyadenylated (APA) to produce mRNAs with different 3' ends that may either alter protein coding sequence (CDS-APA) or create different lengths of 3'UTR (tandem-APA). As the CPA reaction is intimately associated with transcriptional termination, it has been widely assumed that APA is regulated cotranscriptionally. Isoforms terminated at different regions may have distinct RNA stability under different conditions, thus altering the ratio of APA isoforms. Such differential impacts on different isoforms have been considered as post-transcriptional APA, but strictly speaking, this can only be considered "apparent" APA, as the choice is not made during the CPA reaction. Interestingly, a recent study reveals sequential APA as a new mechanism for post-transcriptional APA. This minireview will focus on this new mechanism to provide insights into various documented regulatory paradigms.

Diverse cell-specific patterns of alternative polyadenylation in Drosophila

Most genes in higher eukaryotes express isoforms with distinct 3’ untranslated regions (3’ UTRs), generated by alternative polyadenylation (APA). Since 3’ UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3’-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3’ UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3’ UTR lengthening in differentiated neurons and 3’ UTR shortening in spermatocytes and spermatids. We trace dynamic 3’ UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3’ isoforms across the major cell types of this widely-studied model organism.

Identification of a direct role of CPSF6 and a key cis‐acting motif in promoting TRIM9 proximal polyA site usage

Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3’ termini, is a rapidly expanding area of research that in recent studies has been linked to the mechanistic target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism. Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling and APA remains poorly understood. We previously leveraged our cTag‐PAPERCLIP technique to generate a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin ligase with a role in neurodevelopment, as a gene of interest. In this study, we further characterized the regulation of the mTOR‐induced TRIM9 APA shift observed in mouse neurons in vivo. Further study of the regulation of TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms, with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Additional study into the 3’UTR sequence elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence elements essential for CPSF6‐mediated regulation of the proximal TRIM9 PAS, we developed a RT‐qPCR PAS competition assay to quantify sequence‐mediated usage of PASs. Utilizing this assay, we assessed usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we generated constructs containing mutated UGUA sequences in order to ascertain the importance of the UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the TRIM9 proximal PAS. Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a reduction in TRIM9 proximal PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis‐acting motif in promoting TRIM9 proximal PAS usage. Furthermore, our results also suggest a possible link between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and neurodevelopment through TRIM9.

The Cap-Binding Complex CBC and the Eukaryotic Translation Factor eIF4E: Co-Conspirators in Cap-Dependent RNA Maturation and Translation.

Simple SummaryTo produce the proteins needed for the cell to survive, the information in the DNA is converted to a mobile form known as messenger RNA, which exits the cell nucleus and binds to machines that convert RNAs into proteins in a process referred to as translation. Importantly, the RNA message can be altered at any point in its journey prior to translation. These alterations constitute changes to the chemical nature of the messenger RNA and modulate the ability of mRNAs to be converted into proteins and even lead to the production of proteins with different functionalities than those encoded by their original forms. Here we provide a conceptual framework for the integration of these mRNA maturation steps with translation with a focus on the proteins that escort these mRNAs through these steps and into the translation machines. We discuss the relevance to cancer and therapeutic strategies to target these in malignancy.The translation of RNA into protein is a dynamic process which is heavily regulated during normal cell physiology and can be dysregulated in human malignancies. Its dysregulation can impact selected groups of RNAs, modifying protein levels independently of transcription. Integral to their suitability for translation, RNAs undergo a series of maturation steps including the addition of the m7G cap on the 5′ end of RNAs, splicing, as well as cleavage and polyadenylation (CPA). Importantly, each of these steps can be coopted to modify the transcript signal. Factors that bind the m7G cap escort these RNAs through different steps of maturation and thus govern the physical nature of the final transcript product presented to the translation machinery. Here, we describe these steps and how the major m7G cap-binding factors in mammalian cells, the cap binding complex (CBC) and the eukaryotic translation initiation factor eIF4E, are positioned to chaperone transcripts through RNA maturation, nuclear export, and translation in a transcript-specific manner. To conceptualize a framework for the flow and integration of this genetic information, we discuss RNA maturation models and how these integrate with translation. Finally, we discuss how these processes can be coopted by cancer cells and means to target these in malignancy.

Integrator enforces the fidelity of transcriptional termination at protein-coding genes.

Integrator regulates the 3′-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3′-end processing and termination of a set of protein-coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de novo trimethylation of lysine-36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex beyond the canonical polyadenylation sites. 3′ RNA sequencing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes features downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3′UTR. Together, this study identifies a subset of protein-coding transcripts whose 3′ end processing requires the Integrator complex.

Known sequence features can explain half of all human gene ends.

Cleavage and polyadenylation (CPA) sites define eukaryotic gene ends. CPA sites are associated with five key sequence recognition elements: the upstream UGUA, the polyadenylation signal (PAS), and U-rich sequences; the CA/UA dinucleotide where cleavage occurs; and GU-rich downstream elements (DSEs). Currently, it is not clear whether these sequences are sufficient to delineate CPA sites. Additionally, numerous other sequences and factors have been described, often in the context of promoting alternative CPA sites and preventing cryptic CPA site usage. Here, we dissect the contributions of individual sequence features to CPA using standard discriminative models. We show that models comprised only of the five primary CPA sequence features give highest probability scores to constitutive CPA sites at the ends of coding genes, relative to the entire pre-mRNA sequence, for 41% of all human genes. U1-hybridizing sequences provide a small boost in performance. The addition of all known RBP RNA binding motifs to the model, however, increases this figure to 49%, and suggests an involvement of both known and suspected CPA regulators as well as potential new factors in delineating constitutive CPA sites. To our knowledge, this high effectiveness of established features to predict human gene ends has not previously been documented.

Cleavage and Polyadenylation Specific Factor 1 Promotes Tumor Progression via Alternative Polyadenylation and Splicing in Hepatocellular Carcinoma.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism required for cleavage and polyadenylation (CPA) of the 3′ untranslated region (3′ UTR) of mRNAs. Several aberrant APA events have been reported in hepatocellular carcinoma (HCC). However, the regulatory mechanisms underlying APA remain unclear. In this study, we found that the expression of cleavage and polyadenylation specific factor 1 (CPSF1), a major component of the CPA complex, was significantly increased in HCC tissues and correlated with unfavorable survival outcomes. Knockdown of CPSF1 inhibited HCC cell proliferation and migration, whereas overexpression of CPSF1 caused the opposite effect. Based on integrative analysis of Iso-Seq and RNA-seq data from HepG2.2.15 cells, we identified a series of transcripts with differential 3′ UTR lengths following the knockdown of CPSF1. These transcripts were related to the biological functions of gene transcription, cytoskeleton maintenance, and endomembrane system transportation. Moreover, knockdown of CPSF1 induced an increase in alternative splicing (AS) events in addition to APA. Taken together, this study provides new insights into our understanding of the post-transcriptional regulatory mechanisms in HCC and implies that CPSF1 may be a potential prognostic biomarker and therapeutic target for HCC.

Modulation of alternative cleavage and polyadenylation events by dCas9-mediated CRISPRpas.

The CRISPR/Cas9 technology is revolutionizing genomic engineering. The high efficiency and selectivity of the system have inspired the development of various derived tools for gene regulation at different levels, such as transcriptional activation or inhibition, epigenetic modification, splicing, and base editing. Cleavage and polyadenylation (CPA) is an essential 3' end maturation step for almost all eukaryotic mRNAs. CPA is tightly coupled with transcriptional termination, and its activity impacts gene expression. Over half of all human genes display alternative polyadenylation (APA), where multiple cleavage and polyadenylation sites (PASs) lead to mRNA isoforms with variable termini. APA isoforms often have distinct metabolisms, and their relative abundance can change drastically in different cells. Here, we describe a method based on delivering a catalytically dead Cas9 (dCas9) to genomic regions nears the PAS, which alters APA site usage in 3'UTRs or introns. This method, named CRISPRpas, allows investigators to examine functional significance of APA isoforms of individual genes. We also describe using the bioinformatics program APAlyzer to examine APA events of interest with RNA-seq data.

Integrator is a genome-wide attenuator of non-productive transcription

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.

Emerging Roles of RNA 3'-end Cleavage and Polyadenylation in Pathogenesis, Diagnosis and Therapy of Human Disorders.

A crucial feature of gene expression involves RNA processing to produce 3′ ends through a process termed 3′ end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer.

Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

SUMMARYThe eukaryotic translation initiation factor eIF4E is nuclear and cytoplasmic where it plays roles in export and translation of specific transcripts, respectively. When we were studying its mRNA export activity, we unexpectedly discovered that eIF4E drives the protein expression of elements of the 3′-end core cleavage complex involved in cleavage and polyadenylation (CPA), including CPSF3, the enzyme responsible for cleavage, as well as its co-factors CPSF1, CPSF2, CPSF4, Symplekin, WDR33, and FIP1L1. Using multiple strategies, we demonstrate that eIF4E stimulates 3′-end cleavage of selected RNAs. eIF4E physically interacts with CPSF3, CPSF1, and uncleaved target RNA, suggesting it acts directly and indirectly on the pathway. Through these effects, eIF4E can generate better substrates for its mRNA export and translation activities. Thus, we identified an unanticipated function for eIF4E in 3′-end processing of specific target RNAs, and this function could potentially affect the expression of a broad range of oncoproteins.

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

SUMMARYRegulation 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.

Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination

SummaryThe pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3′ mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.