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Proceedings of the National Academy of... Feb 2018Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the...
Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.
Topics: Cleavage And Polyadenylation Specificity Factor; Cryoelectron Microscopy; Humans; Models, Molecular; Nuclear Proteins; Poly A; Polyadenylation; Protein Conformation; RNA Precursors; RNA, Messenger
PubMed: 29208711
DOI: 10.1073/pnas.1718723115 -
The Journal of Biological Chemistry May 2003The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic...
The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.
Topics: Base Sequence; Chloroplasts; Cyanobacteria; Endoribonucleases; Escherichia coli; Molecular Sequence Data; Poly A; Polynucleotide Adenylyltransferase; Polyribonucleotide Nucleotidyltransferase; RNA, Messenger
PubMed: 12601000
DOI: 10.1074/jbc.M211571200 -
Journal of Virology Feb 2017Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA...
UNLABELLED
Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3' end regulates its capsid expression.
IMPORTANCE
The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2 does not contain typical polyadenylation signals, and the last 42 nt form a stem-loop structure which is almost identical to that of MVC. Further study showed that NS1, REH, and cis elements of (pA)d are required for efficient polyadenylation at (pA)d2. Polyadenylation at (pA)d2 enhances capsid expression. Our study demonstrates alternative polyadenylation at the 3' end of HBoV and suggests an additional mechanism by which capsid expression is regulated.
Topics: Alternative Splicing; Base Sequence; Capsid Proteins; Cell Line; Gene Expression Regulation, Viral; Human bocavirus; Humans; Mutation; Poly A; Polyadenylation; RNA, Messenger; Regulatory Sequences, Nucleic Acid; Terminal Repeat Sequences; Transcription, Genetic
PubMed: 27881651
DOI: 10.1128/JVI.02026-16 -
BMC Bioinformatics 2013Polyadenylation is present in all three domains of life, making it the most conserved post-transcriptional process compared with splicing and 5'-capping. Even though...
BACKGROUND
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.
RESULTS
We propose a poly(A) site model that employs minimal features to capture the essence of poly(A) sites, and yet, produces better prediction accuracy across diverse species. Our model consists of three dior-trinucleotide profiles identified through principle component analysis, and the predicted nucleosome occupancy flanking the poly(A) sites. We validated our model using two machine learning methods: logistic regression and linear discriminant analysis. Results show that models achieve 85-92% sensitivity and 85-96% specificity in seven animals and plants. When we applied one model from one species to predict poly(A) sites from other species, the sensitivity scores correlate with phylogenetic distances.
CONCLUSIONS
A four-feature model geared towards small motifs was sufficient to accurately learn and predict poly(A) sites across eukaryotes.
Topics: Animals; Artificial Intelligence; Conserved Sequence; Discriminant Analysis; Logistic Models; Models, Theoretical; Nucleosomes; Phylogeny; Plants; Poly A; Polyadenylation; Principal Component Analysis; RNA, Messenger; Sensitivity and Specificity
PubMed: 23368518
DOI: 10.1186/1471-2105-14-S2-S9 -
Nucleic Acids Research Jan 2016Cap homeostasis is a cyclical process of decapping and recapping that maintains the cap on a subset of the cytoplasmic transcriptome. Interfering with cytoplasmic...
Cap homeostasis is a cyclical process of decapping and recapping that maintains the cap on a subset of the cytoplasmic transcriptome. Interfering with cytoplasmic capping results in the redistribution of target transcripts from polysomes to non-translating mRNPs, where they accumulate in an uncapped but nonetheless stable form. It is generally thought that decapping is preceded by shortening of the poly(A) tail to a length that can no longer support translation. Therefore recapped target transcripts would either have to undergo cytoplasmic polyadenylation or retain a reasonably long poly(A) tail if they are to return to the translating pool. In cells that are inhibited for cytoplasmic capping there is no change in the overall distribution of poly(A) lengths or in the elution profile of oligo(dT)-bound targets. Poly(A) tail lengths were similar for target mRNAs on polysomes or in non-translating mRNPs, and the presence of polyadenylated uncapped mRNA in mRNPs was confirmed by separation into capped and uncapped pools prior to assay. Finally, in silico analysis of cytoplasmic capping targets revealed significant correlations with genes encoding transcripts with uridylated or multiply modified 3' ends, and genes possessing multiple 3'-untranslated regions (UTRs) generated by alternative cleavage and polyadenylation.
Topics: 3' Untranslated Regions; Animals; Cell Line; Cytoplasm; Homeostasis; Poly A; Polyadenylation; Polyribosomes; Protein Biosynthesis; RNA Caps; RNA, Messenger; RNA, Untranslated; Ribonucleoproteins
PubMed: 26673707
DOI: 10.1093/nar/gkv1460 -
RNA Biology 2016The generation of mature mRNA in the protozoan parasite Trypanosoma brucei requires coupled polyadenylation and trans splicing. In contrast to other eukaryotes, we still...
The generation of mature mRNA in the protozoan parasite Trypanosoma brucei requires coupled polyadenylation and trans splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3' end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in T. brucei, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3' end processing of mRNAs. Based on tandem-affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both in vivo polyadenylation and trans splicing, arguing for a general coupling of these two mRNA-processing reactions.
Topics: DNA-Directed RNA Polymerases; Introns; Poly A; Polyadenylation; RNA, Messenger; Trans-Splicing; Trypanosoma brucei brucei
PubMed: 26727667
DOI: 10.1080/15476286.2015.1130208 -
Cell Reports Mar 2012We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across...
We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.
Topics: 3' Untranslated Regions; Animals; Base Sequence; Blotting, Northern; Conserved Sequence; DNA-Binding Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Genes, Insect; In Situ Hybridization; Male; Molecular Sequence Data; Neurons; Nucleotide Motifs; Organ Specificity; Poly A; Polyadenylation; Protein Isoforms; RNA, Messenger; Reproducibility of Results; Sequence Analysis, RNA; Testis; Transcriptome
PubMed: 22685694
DOI: 10.1016/j.celrep.2012.01.001 -
Nature Communications Sep 2021Deciphering the post-transcriptional mechanisms (PTM) regulating gene expression is critical to understand the dynamics underlying transcriptomic regulation in cancer....
Deciphering the post-transcriptional mechanisms (PTM) regulating gene expression is critical to understand the dynamics underlying transcriptomic regulation in cancer. Alternative polyadenylation (APA)-regulation of mRNA 3'UTR length by alternating poly(A) site usage-is a key PTM mechanism whose comprehensive analysis in cancer remains an important open challenge. Here we use a method and analysis pipeline that sequences 3'end-enriched RNA directly to overcome the saturation limitation of traditional 5'-3' based sequencing. We comprehensively map the APA landscape in lung cancer in a cohort of 98 tumor/non-involved tissues derived from European American and African American patients. We identify a global shortening of 3'UTR transcripts in lung cancer, with notable functional implications on the expression of both coding and noncoding genes. We find that APA of non-coding RNA transcripts (long non-coding RNAs and microRNAs) is a recurrent event in lung cancer and discover that the selection of alternative polyA sites is a form of non-coding RNA expression control. Our results indicate that mRNA transcripts from EAs are two times more likely than AAs to undergo APA in lung cancer. Taken together, our findings comprehensively map and identify the important functional role of alternative polyadenylation in determining transcriptomic heterogeneity in lung cancer.
Topics: 3' Untranslated Regions; Black or African American; Aged; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Poly A; Polyadenylation; Prognosis; RNA, Messenger; RNA, Untranslated; United States; White People
PubMed: 34556645
DOI: 10.1038/s41467-021-25763-5 -
Methods in Molecular Biology (Clifton,... 2014mRNA polyadenylation functions in nuclear export, translation, and stability. We describe an efficient protocol designed to assess poly(A) tail length that is based on...
mRNA polyadenylation functions in nuclear export, translation, and stability. We describe an efficient protocol designed to assess poly(A) tail length that is based on 3' tailing by yeast poly(A) polymerase and product analysis to single-nucleotide resolution by capillary electrophoresis.
Topics: Electrophoresis, Capillary; Genetic Techniques; Poly A; Polynucleotide Adenylyltransferase; RNA, Messenger
PubMed: 24590776
DOI: 10.1007/978-1-62703-971-0_2 -
Genes & Development May 2015The key RNA sequence elements and protein factors necessary for 3' processing of polyadenylated mRNA precursors are well known. Recent studies, however, have... (Review)
Review
The key RNA sequence elements and protein factors necessary for 3' processing of polyadenylated mRNA precursors are well known. Recent studies, however, have significantly reshaped current models for the protein-RNA interactions involved in poly(A) site recognition, painting a picture more complex than previously envisioned and also providing new insights into regulation of this important step in gene expression. Here we review the recent advances in this area and provide a perspective for future studies.
Topics: Amino Acid Motifs; Cleavage And Polyadenylation Specificity Factor; Conserved Sequence; Gene Expression Regulation; Polyadenylation; Protein Binding; RNA Precursors; RNA, Messenger
PubMed: 25934501
DOI: 10.1101/gad.261974.115