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Polyadenylation, an essential step in eukaryotic gene expression, requires both cis-elements and a plethora of trans-acting polyadenylation factors. The polyadenylation factors are largely conserved across mammals and fungi. The conservation seems also extended to plants based on the analyses of Arabidopsis polyadenylation factors. To extend this observation, we systemically identified the orthologs of yeast and human polyadenylation factors from 10 plant species chosen based on both the availability of their genome sequences and their positions in the evolutionary tree, which render them representatives of different plant lineages.
Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
Regulatory elements in the 3' untranslated regions (UTRs) of eukaryotic mRNAs influence mRNA localization, translation, and stability. 3'-UTR length is determined by the location at which mRNAs are cleaved and polyadenylated. The use of alternative polyadenylation sites is common, and can be regulated in different situations. I present a new method to identify cleavage and polyadenylation sites (CSs) at the genome-wide level. The approach is strand-specific, avoids RNA enzymatic modification steps that can introduce sequence-specific biases, and uses unique molecular identifiers to ensure that all identified CS originates from individual RNA molecules. I applied this method to create the first comprehensive genome-wide map of polyadenylation sites of the fission yeast Schizosaccharomyces pombe, comprising the analysis of 2,021,000 individual mRNAs that defined 8,883 CSs. CSs were identified for 90% of coding genes and 50% of ncRNAs. Alternative polyadenylation was prevalent in both groups, with 41% and 45% of all detected genes, respectively, displaying more than one CS. The specificity of the cleavage reaction was gene-specific, resulting in highly variable levels of heterogeneity in 3'-UTR lengths. Finally, I show that for both coding and non-coding genes, the most common regulatory motif associated with CSs in fission yeast is the canonical human AAUAAA sequence.
A biologically important human gene, cyclooxygenase-2 (COX-2), has been proposed to be regulated at many levels. While COX-1 is constitutively expressed in cells, COX-2 is inducible and is upregulated in response to many signals. Since increased transcriptional activity accounts for only part of the upregulation of COX-2, we chose to explore other RNA processing mechanisms in the regulation of this gene. We performed a comprehensive bioinformatics survey, the first of its kind known for human COX-2, which revealed that the human COX-2 gene has alternative polyadenylation (proximal and distal sites) and suggested that use of the alternative polyadenylation signals has tissue specificity. We experimentally established this in HepG2 and HT29 cells. We used an in vivo polyadenylation assay to examine the relative strength of the COX-2 proximal and distal polyadenylation signals, and have shown that the proximal polyadenylation signal is much weaker than the distal one. The efficiency of utilization of many suboptimal mammalian polyadenylation signals is affected by sequence elements located upstream of the AAUAAA, known as upstream efficiency elements (USEs). Here, we used in vivo polyadenylation assays in multiple cell lines to demonstrate that the COX-2 proximal polyadenylation signal contains USEs, mutation of the USEs substantially decreased usage of the proximal signal, and that USE spacing relative to the polyadenylation signal was significant. In addition, mutation of the COX-2 proximal polyadenylation signal to a more optimal sequence enhanced polyadenylation efficiency 3.5-fold. Our data suggest for the first time that alternative polyadenylation of COX-2 is an important post-transcriptional regulatory event.
Translation in axons is required for growth cone chemotropic responses to many guidance cues. Although locally synthesized proteins are beginning to be identified, how specific mRNAs are selected for translation remains unclear. Control of poly(A) tail length by cytoplasmic polyadenylation element (CPE) binding protein 1 (CPEB1) is a conserved mechanism for mRNA-specific translational regulation that could be involved in regulating translation in axons.
Alternative polyadenylation (APA) plays a critical role in regulating gene expression. However, the balance between genome-encoded APA processing and autoregulation by APA modulating RNA binding protein (RBP) factors is not well understood. We discovered two potent small-molecule modulators of APA (T4 and T5) that promote distal-to-proximal (DtoP) APA usage in multiple transcripts. Monotonically responsive APA events, induced by short exposure to T4 or T5, were defined in the transcriptome, allowing clear isolation of the genomic sequence features and RBP motifs associated with DtoP regulation. We found that longer vulnerable introns, enriched with distinctive A-rich motifs, were preferentially affected by DtoP APA, thus defining a core set of genes with genomically encoded DtoP regulation. Through APA response pattern and compound-small interfering RNA epistasis analysis of APA-associated RBP factors, we further demonstrated that DtoP APA usage is partly modulated by altered autoregulation of polyadenylate binding nuclear protein-1 signaling.
Polyadenylation is present in all three domains of life, making it the most conserved post-transcriptional process compared with splicing and 5'-capping. Even though most mammalian poly(A) sites contain a highly conserved hexanucleotide in the upstream region and a far less conserved U/GU-rich sequence in the downstream region, there are many exceptions. Furthermore, poly(A) sites in other species, such as plants and invertebrates, exhibit high deviation from this genomic structure, making the construction of a general poly(A) site recognition model challenging. We surveyed nine poly(A) site prediction methods published between 1999 and 2011. All methods exploit the skewed nucleotide profile across the poly(A) sites, and the highly conserved poly(A) signal as the primary features for recognition. These methods typically use a large number of features, which increases the dimensionality of the models to crippling degrees, and typically are not validated against many kinds of genomes.
Alternative polyadenylation is one of the mechanisms in human cells that give rise to a variety of transcripts from a single gene. More than half of the human genes have multiple polyadenylation sites (poly(A) sites), leading to variable mRNA and protein products. Previous studies of individual genes have indicated that alternative polyadenylation could occur in a tissue-specific manner.
Processing of transcripts at the 3'-end involves cleavage at a polyadenylation site followed by the addition of a poly(A)-tail. By selecting which site is cleaved, the process of alternative polyadenylation enables genes to produce transcript isoforms with different 3'-ends. To facilitate the identification and treatment of disease-causing mutations that affect polyadenylation and to understand the sequence determinants underlying this regulatory process, a computational model that can accurately predict polyadenylation patterns from genomic features is desirable.
The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such template-switching artifacts on alternative polyadenylation are generally disregarded, while alternative polyadenylation artifacts are attributed to internal priming.
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.
Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3' ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight.
The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding of polyadenylation-mediated regulation of gene expression within the DNA sequence is not well understood. Here, we utilized a massively parallel reporter assay to inspect the effect of over 12,000 rationally designed polyadenylation sequences (PASs) on reporter gene expression and cleavage efficiency. We find that the PAS sequence can modulate gene expression by over five orders of magnitude. By using a uniquely designed scanning mutagenesis data set, we gain mechanistic insight into various modes of action by which the cleavage efficiency affects the sensitivity or robustness of the PAS to mutation. Furthermore, we employ motif discovery to identify both known and novel sequence motifs associated with PAS-mediated regulation. By leveraging the large scale of our data, we train a deep learning model for the highly accurate prediction of RNA levels from DNA sequence alone (R = 0.83). Moreover, we devise unique approaches for predicting exact cleavage sites for our reporter constructs and for endogenous transcripts. Taken together, our results expand our understanding of PAS-mediated regulation, and provide an unprecedented resource for analyzing and predicting PAS for regulatory genomics applications.
Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals.
The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT-PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.
Messenger RNA polyadenylation, the process wherein the primary RNA polymerase II transcript is cleaved and a poly(A) tract added, is a key step in the expression of genes in plants. Moreover, it is a point at which gene expression may be regulated by determining the functionality of the mature mRNA. Polyadenylation is mediated by a complex (the polyadenylation complex, or PAC) that consists of between 15 and 20 subunits. While the general functioning of these subunits may be inferred by extending paradigms established in well-developed eukaryotic models, much remains to be learned about the roles of individual subunits in the regulation of polyadenylation in plants. To gain further insight into this, we conducted a survey of variability in the plant PAC. For this, we drew upon a database of naturally-occurring variation in numerous geographic isolates of Arabidopsis thaliana. For a subset of genes encoding PAC subunits, the patterns of variability included the occurrence of premature stop codons in some Arabidopsis accessions. These and other observations lead us to conclude that some genes purported to encode PAC subunits in Arabidopsis are actually pseudogenes, and that others may encode proteins with dispensable functions in the plant. Many subunits of the PAC showed patterns of variability that were consistent with their roles as essential proteins in the cell. Several other PAC subunits exhibit patterns of variability consistent with selection for new or altered function. We propose that these latter subunits participate in regulatory interactions important for differential usage of poly(A) sites.
Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15-20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two-poly(A) polymerase and CPSF73-of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages-extracellular merozoites and intracellular schizonts-studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.
Genes containing multiple pre-mRNA cleavage and polyadenylation sites, or polyA sites, express mRNA isoforms with variable 3' untranslated regions (UTRs). By systematic analysis of human and mouse transcriptomes, we found that short 3'UTR isoforms are relatively more abundant when genes are highly expressed whereas long 3'UTR isoforms are relatively more abundant when genes are lowly expressed. Reporter assays indicated that polyA site choice can be modulated by transcriptional activity through the gene promoter. Using global and reporter-based nuclear run-on assays, we found that RNA polymerase II is more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, highly expressed genes tend to have a lower level of nucleosome but higher H3K4me3 and H3K36me3 levels at promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that polyA site usage is generally coupled to transcriptional activity, leading to regulation of alternative polyadenylation by transcription.
The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown.
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