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Genome Biology Jul 2012The 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...
BACKGROUND
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.
RESULTS
Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation.
CONCLUSIONS
Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.
Topics: 3' Untranslated Regions; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; E2F Transcription Factors; Fibroblasts; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Humans; Poly A; Polyadenylation; Sequence Analysis, RNA
PubMed: 22747694
DOI: 10.1186/gb-2012-13-7-r59 -
Cell Chemical Biology Dec 2018Alternative polyadenylation (APA) plays a critical role in regulating gene expression. However, the balance between genome-encoded APA processing and autoregulation by...
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.
Topics: Cell Line; Female; Homeostasis; Humans; Polyadenylation; Small Molecule Libraries; Transcriptome
PubMed: 30293940
DOI: 10.1016/j.chembiol.2018.09.006 -
Nucleic Acids Research Jun 1988We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein...
We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing.
Topics: Base Sequence; Cell Nucleus; HeLa Cells; Humans; Plasmids; Poly A; Protein Binding; RNA Precursors; RNA, Messenger; Simplexvirus
PubMed: 2898767
DOI: 10.1093/nar/16.12.5323 -
The Plant Journal : For Cell and... Jan 2018Auxin is widely involved in plant growth and development. However, the molecular mechanism on how auxin carries out this work is unclear. In particular, the effect of...
Auxin is widely involved in plant growth and development. However, the molecular mechanism on how auxin carries out this work is unclear. In particular, the effect of auxin on pre-mRNA post-transcriptional regulation is mostly unknown. By using a poly(A) tag (PAT) sequencing approach, mRNA alternative polyadenylation (APA) profiles after auxin treatment were revealed. We showed that hundreds of poly(A) site clusters (PACs) are affected by auxin at the transcriptome level, where auxin reduces PAC distribution in 5'-untranslated region (UTR), but increases in the 3'UTR. APA site usage frequencies of 42 genes were switched by auxin, suggesting that auxin affects the choice of poly(A) sites. Furthermore, poly(A) signal selection was altered after auxin treatment. For example, a mutant of poly(A) signal binding protein CPSF30 showed altered sensitivity to auxin treatment, indicating interactions between auxin and the poly(A) signal recognition machinery. We also found that auxin activity on lateral root development is likely mediated by altered expression of ARF7, ARF19 and IAA14 through poly(A) site switches. Our results shed light on the molecular mechanisms of auxin responses relative to its interactions with mRNA polyadenylation.
Topics: Arabidopsis; Arabidopsis Proteins; Flowers; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Indoleacetic Acids; Plant Growth Regulators; Plant Roots; Poly A; Polyadenylation; RNA, Messenger; RNA, Plant
PubMed: 29155478
DOI: 10.1111/tpj.13771 -
BMC Genomics Jul 2022Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA,...
BACKGROUND
Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.
RESULTS
Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.
CONCLUSIONS
Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.
Topics: High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; RNA; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 35869428
DOI: 10.1186/s12864-022-08762-8 -
International Journal of Molecular... Sep 2021Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3'-terminus of many mammalian SINEs...
Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3'-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.
Topics: Animals; Evolution, Molecular; Humans; Mice; Poly A; Polyadenylation; RNA; RNA 3' Polyadenylation Signals; RNA Polymerase III; RNA, Messenger; Retroelements; Short Interspersed Nucleotide Elements; Transcription Termination, Genetic; Transcription, Genetic
PubMed: 34576060
DOI: 10.3390/ijms22189897 -
The Journal of Biological Chemistry May 2019Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3....
Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.
Topics: Humans; Peptide Chain Termination, Translational; Peptide Initiation Factors; Peptide Termination Factors; Poly A; RNA, Messenger; RNA-Binding Proteins; Repressor Proteins
PubMed: 30992367
DOI: 10.1074/jbc.RA118.006856 -
Nucleic Acids Research Mar 2003Primary, secondary and higher-order structures of downstream elements of mammalian pre-mRNA polyadenylation signals [poly(A) signals] are re viewed. We have carried out... (Review)
Review
Primary, secondary and higher-order structures of downstream elements of mammalian pre-mRNA polyadenylation signals [poly(A) signals] are re viewed. We have carried out a detailed analysis on our database of 244 human pre-mRNA poly(A) signals in order to characterize elements in their downstream regions. We suggest that the downstream region of the mammalian pre-mRNA poly(A) signal consists of various simple elements located at different distances from each other. Thus, the downstream region is not described by any precise consensus. Searching our database, we found that approximately 80% of pre-mRNAs with the AAUAAA or AUUAAA core upstream elements contain simple downstream elements, consisting of U-rich and/or 2GU/U tracts, the former occurring approximately 2-fold more often than the latter. Approximately one-third of the pre-mRNAs analyzed here contain sequences that may form G-quadruplexes. A substantial number of these sequences are located immediately downstream of the poly(A) signal. A possible role of G-rich sequences in the polyadenylation process is discussed. A model of the secondary structure of the SV40 late pre-mRNA poly(A) signal downstream region is presented.
Topics: Animals; Base Sequence; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Poly A; RNA Precursors; Signal Transduction
PubMed: 12595544
DOI: 10.1093/nar/gkg241 -
ELife Jul 2021Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic...
Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.
Topics: 3' Untranslated Regions; DNA Helicases; Kinetics; Poly A; Polyadenylation; RNA Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34232857
DOI: 10.7554/eLife.65331 -
G3 (Bethesda, Md.) Jun 2019Poly(A)-tail targeted RNAseq approaches, such as 3'READS, PAS-Seq and Poly(A)-ClickSeq, are becoming popular alternatives to random-primed RNAseq to focus sequencing...
Poly(A)-tail targeted RNAseq approaches, such as 3'READS, PAS-Seq and Poly(A)-ClickSeq, are becoming popular alternatives to random-primed RNAseq to focus sequencing reads just to the 3' ends of polyadenylated RNAs to identify poly(A)-sites and characterize changes in their usage. Additionally, we and others have demonstrated that these approaches perform similarly to other RNAseq strategies for differential gene expression analysis, while saving on the volume of sequencing data required and providing a simpler library synthesis strategy. Here, we present DPAC ( ifferential oly( )- lustering); a streamlined pipeline for the preprocessing of poly(A)-tail targeted RNAseq data, mapping of poly(A)-sites, poly(A)-site clustering and annotation, and determination of differential poly(A)-cluster usage using DESeq2. Changes in poly(A)-cluster usage is simultaneously used to report differential gene expression, differential terminal exon usage and alternative polyadenylation (APA).
Topics: Biomarkers; Cluster Analysis; Computational Biology; Exons; Gene Expression Regulation; Gene Knockdown Techniques; HeLa Cells; Humans; Molecular Sequence Annotation; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA; Software
PubMed: 31023725
DOI: 10.1534/g3.119.400273