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![Schematic drawing of the eukaryotic 3′ end processing machineries. Known factors and cis-elements contributing to 3′ end processing of metazoan (A), yeast (B) and plant (C) pre-mRNAs, as described in the text. Homologous factors are color-matched while specific factors are in gray. The position of the different factors takes into account the RNA-binding specificity of each factor and, where possible, the protein contacts within the machinery. The sequence elements that comprise the poly(A) signals are indicated by black rectangles, and the site of cleavage [and subsequent poly(A) tail addition] is shown by a red dotted-line. In (C) ‘At’ stands for Arabidopsis thaliana.](publication/40821585/figure/fig2/AS:213967684280325@1428025279701/Schematic-drawing-of-the-eukaryotic-3-end-processing-machineries-Known-factors-and.png)
Schematic drawing of the eukaryotic 3′ end processing machineries. Known factors and cis-elements contributing to 3′ end processing of metazoan (A), yeast (B) and plant (C) pre-mRNAs, as described in the text. Homologous factors are color-matched while specific factors are in gray. The position of the different factors takes into account the RNA-binding specificity of each factor and, where possible, the protein contacts within the machinery. The sequence elements that comprise the poly(A) signals are indicated by black rectangles, and the site of cleavage [and subsequent poly(A) tail addition] is shown by a red dotted-line. In (C) ‘At’ stands for Arabidopsis thaliana.
Source publication
Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically
cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals
of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene...
Contexts in source publication
Context 1
... 3 0 end processing reaction requires multiple protein factors that are generally conserved in eukaryotes and assemble onto defined sequence elements within the 3 0 end region of the pre-mRNA. Although the cis-elements differ in sequence and location among mammalian, yeast and plant pre-mRNAs, there appears to exist a common tripartite arrangement in which the cleavage site is associated with one A-rich element and one or more U-rich regions (Figure 2). ...Context 2
... machinery leading to the formation of metazoan polyadenylated mRNAs contains several sub-complexes [ Figure 2A, for a recent review see ref. 26], including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CFIm), cleavage factor II (CFIIm), poly(A) polymerase (PAP), symplekin and the pol II. All these factors contrib- ute to the cleavage reaction. ...Context 3
... polyadenylated mRNAs, histone pre-mRNAs 3 0 end processing is governed by a set of rigid constraints that allow a precise coordination between regulation of their expression and DNA replication signals (Figure 2A; for recent reviews see refs 28 and 52). The replication-dependent histone processing signal lies within 100 nt downstream of the stop codon and is composed of a conserved stem-loop sequence and a more variable purine-rich element (histone downstream element or HDE) that begins 15-20 nt downstream of the stem-loop. ...Context 4
... factors comprised in the 3 0 end processing apparatus in mammals and in yeast are generally conserved but the poly(A) signals in the two organisms are rather different in consensus sequence and organization ( Figure 2B; for a recent review see ref. 26). The yeast machinery comprises the cleavage and polyadenylation factor (CPF), the cleavage factor IA (CFIA) and the cleavage factor IB (CFIB). ...Context 5
... human/yeast genes have homologs in plant Arabidopsis genome, except for the mammalian factor CFI (absent also in yeast) and the yeast factor Hrp1 ( Figure 2C; for a recent review see ref. 27). The plant cleavage/polyadenylation machinery is still forthcoming but a working model starts to emerge. ...Context 6
... over the past 20 years contributed to better char- acterize the constitutive factors of the basal eukaryotic 3 0 end processing machineries (depicted in Figure 2) and to identify many other factors that modulate the efficiency and specificity of poly(A) signal recognition by these machineries (Table 1). For some of these factors, the mechanisms underlying the regulatory function have been described and overlapping models of regulation can be proposed (Figure 3 and Supplementary Table S1). ...Similar publications
Through alternative polyadenylation, human mRNAs acquire longer or shorter 3′ untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3′ end processing factors CP...
Citations
... The canonical PAS (AAU AAA ) binds to RBPs in the polyadenylation complex as part of constitutive mRNA metabolism [38]. We examined T cell lineage-defining transcripts with well-resolved GCLiPP profiles (due to their high expression levels), including Cd3g (Fig. 4D), Cd3e, Cd4, and Cd8b1 (Additional file 1: Fig. S4). ...
GCLiPP is a global RNA interactome capture method that detects RNA-binding protein (RBP) occupancy transcriptome-wide. GCLiPP maps RBP-occupied sites at a higher resolution than phase separation-based techniques. GCLiPP sequence tags correspond with known RBP binding sites and are enriched for sites detected by RBP-specific crosslinking immunoprecipitation (CLIP) for abundant cytosolic RBPs. Comparison of human Jurkat T cells and mouse primary T cells uncovers shared peaks of GCLiPP signal across homologous regions of human and mouse 3′ UTRs, including a conserved mRNA-destabilizing cis-regulatory element. GCLiPP signal overlapping with immune-related SNPs uncovers stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13059-023-03125-2.
... Another form of RNA processing is polyadenylation, and its distribution in the 5untranslated region (UTR) and 3 -UTR is responsible for the stability of mature transcripts and influences their export to the cytoplasm, their subcellular localization, and recognition by the translational machinery [23,101]. A poly(A) tag sequencing approach showed that multiple alternative polyadenylations were detected in TAA1/TAR and YUC genes; however, it remains unknown whether these alternative polyadenylations are involved in the post-transcriptional regulation of genes related to auxin biosynthesis [23]. ...
The indole-3-pyruvic acid (IPA) pathway is the main auxin biosynthesis pathway in the plant kingdom. Local control of auxin biosynthesis through this pathway regulates plant growth and development and the responses to biotic and abiotic stresses. During the past decades, genetic, physiological, biochemical, and molecular studies have greatly advanced our understanding of tryptophan-dependent auxin biosynthesis. The IPA pathway includes two steps: Trp is converted to IPA by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS/TRYPTOPHAN AMINOTRANSFERASE RELATED PROTEINs (TAA1/TARs), and then IPA is converted to IAA by the flavin monooxygenases (YUCCAs). The IPA pathway is regulated at multiple levels, including transcriptional and post-transcriptional regulation, protein modification, and feedback regulation, resulting in changes in gene transcription, enzyme activity and protein localization. Ongoing research indicates that tissue-specific DNA methylation and miRNA-directed regulation of transcription factors may also play key roles in the precise regulation of IPA-dependent auxin biosynthesis in plants. This review will mainly summarize the regulatory mechanisms of the IPA pathway and address the many unresolved questions regarding this auxin biosynthesis pathway in plants.
... Polyadenylation of mRNA molecules by poly(A) polymerases (PAPS) (Wahle, 1991) is crucial for mRNA stability, export from the nucleus, and efficient translation (Dower et al., 2004;Ford et al., 1997;Fuke & Ohno, 2008;Sachs et al., 1997;Wickens et al., 1997;Wilusz et al., 2001;Wilusz & Wilusz, 2004;Wormington et al., 1996). The polyadenylation machinery includes the multi-protein complexes Cleavage Stimulation Factor and Cleavage and Polyadenylation Specificity Factor, as well as PAPS (Eckmann et al., 2011;Hunt & Messenger, 2008;Millevoi & Vagner, 2010). Canonical PAPS possess both a conserved N-terminal and a less conserved C-terminal domain. ...
... Canonical PAPS possess both a conserved N-terminal and a less conserved C-terminal domain. The N-terminal domain fulfills the catalytic function, whereas the Cterminal domain serves to regulate the activity of canonical PAPS through modification sites and protein interaction domains (Addepalli et al., 2004;Millevoi & Vagner, 2010;Yang et al., 2014). ...
Polyadenylation of mRNAs is critical for their export from the nucleus, stability and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared to in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A)-tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.
... This usually consists of cleavage and polyadenylation steps. USE (upstream element), PAS (polyadenylation signal), DSE (downstream element), and GRS (G-rich sequence) are four known conserved elements used in synthesizing the poly(A) tail, but numerous complexes such as CPSF (cleavage and polyadenylation specificity factor), CSTF (cleavage stimulation factor), CFIm and CFIIm (mammalian cleavage factors 1 and 2), and HCC (histone pre-mRNA cleavage complex) are also involved in the first step, and then recruit cPAP (canonical Poly(A) polymerase) to fulfill the second step in mammals [23][24][25][26]. ...
In eukaryotes, mRNA metabolism requires a sophisticated signaling system. Recent studies have suggested that polyadenylate tail may play a vital role in such a system. The poly(A) tail used to be regarded as a common modification at the 3 end of mRNA, but it is now known to be more than just that. It appears to act as a platform or hub that can be understood in two ways. On the one hand, polyadenylation and deadenylation machinery constantly regulates its dynamic activity; on the other hand, it exhibits the ability to recruit RNA-binding proteins and then interact with diverse factors to send various signals to regulate mRNA metabolism. In this paper, we outline the main complexes that regulate the dynamic activities of poly(A) tails, explain how these complexes participate polyadenylation/deadenylation process and summarize the diverse signals this hub emit. We are trying to make a point that the poly(A) tail can metaphorically act as a "flagman" who is supervised by polyadenylation and deadenylation and sends out signals to regulate the orderly functioning of mRNA metabolism.
... The main mechanism of pre-mRNA 3 0 -end processing is cleavage and polyadenylation (CPA), which involves endonucleolytic cleavage of newly synthesized transcripts and the addition of adenosine residues constituting the poly(A) tail to the generated 3 0 -end. CPA is crucial for mRNA stability, transport to the cytoplasm, and translation (Millevoi & Vagner, 2010;Shi & Manley, 2015). This nuclear process involves the recognition of cis-acting elements in the pre-mRNA by a complex machinery comprising more than 80 proteins (Shi et al, 2009). ...
The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.
... In the processing of the 3' termini, the 3' end of nascent mRNA is cleaved, followed by addition of a poly (A) tail (i.e., polyadenylation) (28). Both cleavage and polyadenylation occur at polyadenylation sites (PASs) which are located within the 3' untranslated regions (3' UTRs), introns, or internal exons (29,30). Most eukaryotic genes contain multiple PASs. ...
Alternative polyadenylation (APA) is a molecular process that generates diversity at the 3’ end of RNA polymerase II transcripts from over 60% of human genes. APA and microRNA regulation are both mechanisms of post-transcriptional regulation of gene expression. As a key molecular mechanism, Alternative polyadenylation often results in mRNA isoforms with the same coding sequence but different lengths of 3’ UTRs, while microRNAs regulate gene expression by binding to specific mRNA 3’ UTRs. Nudix Hydrolase 21 (NUDT21) is a crucial mediator involved in alternative polyadenylation (APA). Different studies have reported a dual role of NUDT21 in cancer (both oncogenic and tumor suppressor). The present review focuses on the functions of APA, miRNA and their interaction and roles in development of different types of tumors.NUDT21 mediated 3’ UTR-APA changes can be used to generate specific signatures that can be used as potential biomarkers in development and disease. Due to the emerging role of NUDT21 as a regulator of the aforementioned RNA processing events, modulation of NUDT21 levels may be a novel viable therapeutic approach.
... These processes are mediated by several ribonucleoproteins and are tightly regulated to ensure that correctly matured mRNAs are facilitated to be exported from the nucleus. However, many aspects of this sophisticated regulation of mRNA processing remain unelucidated [1][2][3]. Recently, the regulatory mechanism of mRNA splicing in the nucleus has been elucidated using splicing inhibitors, revealing that spliceostatin A, pladienolide B, Gex1A, and H3B-7700 inhibit splicing by binding to SF3B1, a component of U2 small nuclear RNA (U2snRNP), which is a spliceosomal factor, contributing to the understanding of splicing regulation [4][5][6][7]. Additionally, aberrant splicing has been reported as the pathogenesis of various diseases, including cancer [8]. ...
Intracellular and intercellular signalling networks play an essential role in optimizing cellular homoeostasis and are thought to be partly reflected in nuclear mRNA dynamics. However, the regulation of nuclear mRNA dynamics by intracellular and intercellular signals remains largely unexplored, and research tools are lacking. Through an original screening based on the mRNA metabolic mechanism, we discovered that eight well-known inhibitors cause significant nuclear poly(A)⁺ RNA accumulation. Among these inhibitors, we discovered a new mRNA metabolic response in which the addition of antimycin A, an inhibitor of mitochondrial respiratory-chain complex III (complex III), resulted in a marked accumulation of poly(A)⁺ RNA near the nuclear speckles. Furthermore, dihydroorotate dehydrogenase (DHODH) inhibitors, a rate-limiting enzyme in the intracellular de novo pyrimidine synthesis reaction that specifically exchanges electrons with complex III, also caused a remarkable accumulation of nuclear poly(A)⁺ RNA adjacent to the nuclear speckles, which was abolished by extracellular uridine supply, indicating that the depletion of intracellular pyrimidine affects poly(A)⁺ RNA metabolism. Further analysis revealed that ataxia telangiectasia mutated (ATM), a serine and threonine kinase and a master regulator of DNA double-strand break (DSB) and nucleolar stress, is required for this poly(A)⁺ RNA nuclear accumulation phenomenon. This study reports new insights into novel aspects of nuclear poly(A)⁺ RNA metabolism, especially the relationship between mitochondrial respiratory-chain functions, pyrimidine metabolism, and nuclear RNA metabolism.
... This factor-dependent transcription termination in VACV is thought to be facilitated by the H5 protein for intermediate and late gene transcripts188,333 . Possibly mirroring the activities of cleavage and polyadenylation specificity factor (CPSF) with cleavage stimulatory factor (CstF), in recognising the PAS and promoting cleavage[334][335][336] . H5 appears to act in concert with further viral factors, since proteins G2 and A18 interact with H5 in vivo337 , with the role of transcript release during termination falling on the ATP-dependent DNA helicase A18 ...
African Swine Fever Virus (ASFV) causes lethal haemorrhagic fever in domestic pigs, presenting the largest global threat to animal farming on record. Despite its impact, the mechanisms and regulation of ASFV gene expression were poorly understood. In order to fill this gap in our knowledge, I have investigated diverse aspects of ASFV transcription and report in my thesis (i) transcriptome analyses, (ii) the expression, purification and biochemical analyses of recombinant transcription factors, and (iii) computational characterisation of ASFV-RNA polymerase (RNAP) subunits and transcription initiation factors. We have generated the first genome-wide transcriptomic landscape of ASFV during infection, using a complement of RNA-based Next Generation Sequencing techniques. We have mapped the ASFV gene transcription start sites, termination sites, and quantified transcript abundance during the early and late stages of infection. We have demonstrated viral gene expression patterns, which are facilitated by newly identified promoter motifs, and shared across lab-attenuated and pathogenic strains (BA71V and Georgia 2007/1, respectively). We have also demonstrated ASFV uses a polyT (polyU in the RNA) terminator motif genome-wide, and delved into how late infection alters transcription termination patterns. We identified a conserved early promoter motif in ASFV, similar to that used by the heterodimeric VACV early transcription factor (VETF), for which ASFV also encodes homologs. We therefore, co-expressed and purified the ASFV VETF subunits (D6 and A7) recombinantly, using a baculovirus-insect cell expression system. We demonstrated this large (284 kDa) ASFV-D6-A7 complex specifically binds to early but not late promoter templates, though with some differences to its VACV counterpart, likely due to the ASFV proteins encoding additional domains. We have therefore demonstrated that baculovirus-insect cell expression is viable for co-expressing large ASFV complexes, and developed the ground work to apply this system to express 8-subunit ASFV-RNAP.
... The mammalian 3 end processing apparatus is composed of 16 'core' proteins associated into the CPSF, CstF, CFIm and CFIIm complexes and a poly(A) polymerase (14,15) and molecular architectures of some of the cleavage and polyadenylation machinery has been detailed (16). The location of pre-mRNA 3 end is mainly defined by consensus poly(A) signals defined by the canonical sequence AAUAAA (17), and the cleavage site ∼15 nt downstream, where polyadenylation occurs. ...
Precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3′UTR. We previously reported the important diversity of 3′UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.
... In our previous study, after screening 21 compounds with a flavone skeleton, we found that apigenin and another compound, luteolin, were able to modulate messenger RNA (mRNA) splicing at the genome-wide level and reduce the viability of several cancer cell lines [34]. mRNA processing (5 capping, splicing, and 3 end processing) is an essential step in sustaining various cell functions, such as proliferation, survival, and differentiation [35][36][37], and causing various pathological conditions, such as the onset and progression of carcinoma [38,39]. The potency of apigenin itself may not be strong, but we expected that it would be able to prevent carcinogenesis by acting for an extended period of time. ...
Oral leukoplakia, which presents as white lesions in the oral cavity, including on the tongue, is precancerous in nature. Conservative treatment is preferable, since surgical removal can markedly reduce the patient’s quality of life. In the present study, we focused on the flavonoid apigenin as a potential compound for preventing carcinogenesis, and an apigenin-loaded mucoadhesive oral film was prepared using a three-dimensional (3D) bioprinter (semi-solid extrusion-type 3D printer). Apigenin-loaded printer inks are composed of pharmaceutical excipients (HPMC, CARBOPOL, and Poloxamer), water, and ethanol to dissolve apigenin, and the appropriate viscosity of printer ink after adjusting the ratios allowed for the successful 3D printing of the film. After drying the 3D-printed object, the resulting film was characterized. The chemopreventive effect of the apigenin-loaded film was evaluated using an experimental rat model that had been exposed to 4-nitroquinoline 1-oxide (4NQO) to induce oral carcinogenesis. Treatment with the apigenin-loaded film showed a remarkable chemopreventive effect based on an analysis of the specimen by immunohistostaining. These results suggest that the apigenin-loaded mucoadhesive film may help prevent carcinogenesis. This successful preparation of apigenin-loaded films by a 3D printer provides useful information for automatically fabricating other tailored films (with individual doses and shapes) for patients with oral leukoplakia in a future clinical setting.
