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RNA (New York, N.Y.) Dec 2000In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during... (Comparative Study)
Comparative Study
In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.
Topics: 5' Untranslated Regions; Cell-Free System; Encephalomyocarditis virus; Gene Expression Regulation, Viral; HeLa Cells; Hepatovirus; Humans; Peptide Chain Initiation, Translational; Picornaviridae; Poliovirus; Poly A; RNA Caps; RNA, Messenger; RNA, Viral; Ribosomes
PubMed: 11142378
DOI: 10.1017/s1355838200001679 -
Biochemistry Nov 2020Influenza A virus (IAV) is a highly contagious human pathogen that is responsible for tens of thousands of deaths each year. Non-structural protein 1 (NS1) is a crucial...
Influenza A virus (IAV) is a highly contagious human pathogen that is responsible for tens of thousands of deaths each year. Non-structural protein 1 (NS1) is a crucial protein expressed by IAV to evade the host immune system. Additionally, NS1 has been proposed to stimulate translation because of its ability to bind poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G. We analyzed the interaction of NS1 with PABP1 using quantitative techniques. Our studies show that NS1 binds as a homodimer to PABP1, and this interaction is conserved across different IAV strains. Unexpectedly, NS1 does not bind to PABP1 that is bound to poly(A) RNA. Instead, NS1 binds only to PABP1 free of RNA, suggesting that stimulation of translation does not occur by NS1 interacting with the PABP1 molecule attached to the mRNA 3'-poly(A) tail. These results suggest that the function of the NS1·PABP1 complex appears to be distinct from the classical role of PABP1 in translation initiation, when it is bound to the 3'-poly(A) tail of mRNA.
Topics: Binding Sites; Electrophoretic Mobility Shift Assay; Fluorescence Polarization; Host-Pathogen Interactions; Influenza A virus; Mutation; Poly A; Poly(A)-Binding Protein I; Protein Multimerization; RNA, Messenger; Static Electricity; Viral Nonstructural Proteins
PubMed: 33172261
DOI: 10.1021/acs.biochem.0c00666 -
PloS One 2017RNA-seq reads containing part of the poly(A) tail of transcripts (denoted as poly(A) reads) provide the most direct evidence for the position of poly(A) sites in the...
RNA-seq reads containing part of the poly(A) tail of transcripts (denoted as poly(A) reads) provide the most direct evidence for the position of poly(A) sites in the genome. However, due to reduced coverage of poly(A) tails by reads, poly(A) reads are not routinely identified during RNA-seq mapping. Nevertheless, recent studies for several herpesviruses successfully employed mapping of poly(A) reads to identify herpesvirus poly(A) sites using different strategies and customized programs. To more easily allow such analyses without requiring additional programs, we integrated poly(A) read mapping and prediction of poly(A) sites into our RNA-seq mapping program ContextMap 2. The implemented approach essentially generalizes previously used poly(A) read mapping approaches and combines them with the context-based approach of ContextMap 2 to take into account information provided by other reads aligned to the same location. Poly(A) read mapping using ContextMap 2 was evaluated on real-life data from the ENCODE project and compared against a competing approach based on transcriptome assembly (KLEAT). This showed high positive predictive value for our approach, evidenced also by the presence of poly(A) signals, and considerably lower runtime than KLEAT. Although sensitivity is low for both methods, we show that this is in part due to a high extent of spurious results in the gold standard set derived from RNA-PET data. Sensitivity improves for poly(A) sites of known transcripts or determined with a more specific poly(A) sequencing protocol and increases with read coverage on transcript ends. Finally, we illustrate the usefulness of the approach in a high read coverage scenario by a re-analysis of published data for herpes simplex virus 1. Thus, with current trends towards increasing sequencing depth and read length, poly(A) read mapping will prove to be increasingly useful and can now be performed automatically during RNA-seq mapping with ContextMap 2.
Topics: Animals; Humans; Poly A; RNA, Messenger; Sequence Analysis, RNA; Software; Transcriptome
PubMed: 28135292
DOI: 10.1371/journal.pone.0170914 -
Frontiers in Bioscience (Landmark... Jan 2009Bovine papillomavirus type 1 (BPV-1) has served as a prototype for studying the molecular biology and pathogenesis of papillomaviruses. The expression of BPV-1 early and... (Review)
Review
Bovine papillomavirus type 1 (BPV-1) has served as a prototype for studying the molecular biology and pathogenesis of papillomaviruses. The expression of BPV-1 early and late genes is highly regulated at both transcription and post-transcriptional levels and strictly tied to the differentiation of keratinocytes. BPV-1 infects keratinocytes in the basal layer of the skin and replicates in the nucleus of infected cells in a differentiation-dependent manner. Although viral early genes begin to be expressed from the infected, undifferentiated basal cells, viral late genes are not expressed until the infected cells enter the terminal differentiation stage. Both BPV-1 early and late transcripts are intron-containing bicistronic or polycistronic RNAs, bearing more than one open reading frame and are polyadenylated at either an early or late poly (A) site. Nuclear RNA processing of these transcripts by RNA splicing and poly (A) site selection has been extensively analyzed in the past decade and various viral cis-elements and cellular factors involved in regulation of viral RNA processing were discovered, leading to our better understanding of the gene expression and biology of human papillomaviruses.
Topics: Bovine papillomavirus 1; Gene Expression Regulation, Viral; Poly A; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Viral
PubMed: 19273129
DOI: 10.2741/3307 -
Nucleic Acids Research Feb 2023RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition...
RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.
Topics: Cold-Shock Response; DNA-Binding Proteins; Poly A; Poly(A)-Binding Proteins; Protein Binding; RNA
PubMed: 36688322
DOI: 10.1093/nar/gkac1277 -
Frontiers in Immunology 2023Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the...
Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3' UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.
Topics: RNA, Messenger; Poly A; Polyadenylation; Cell Differentiation; Macrophages
PubMed: 36761770
DOI: 10.3389/fimmu.2023.1091403 -
International Journal of Molecular... Dec 2019The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive...
The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive secondary species such as low-energy electrons (LEEs) with the most probable energy around 10 eV are generated, which are able to induce DNA strand breaks via dissociative electron attachment. Absolute DNA strand break cross sections of specific DNA sequences can be efficiently determined using DNA origami nanostructures as platforms exposing the target sequences towards LEEs. In this paper, we systematically study the effect of the oligonucleotide length on the strand break cross section at various irradiation energies. The present work focuses on poly-adenine sequences (d(A), d(A), d(A), d(A), and d(A)) irradiated with 5.0, 7.0, 8.4, and 10 eV electrons. Independent of the DNA length, the strand break cross section shows a maximum around 7.0 eV electron energy for all investigated oligonucleotides confirming that strand breakage occurs through the initial formation of negative ion resonances. When going from d(A) to d(A), the strand break cross section increases with oligonucleotide length, but only at 7.0 and 8.4 eV, i.e., close to the maximum of the negative ion resonance, the increase in the strand break cross section with the length is similar to the increase of an estimated geometrical cross section. For d(A), a markedly lower DNA strand break cross section is observed for all electron energies, which is tentatively ascribed to a conformational change of the dA sequence. The results indicate that, although there is a general length dependence of strand break cross sections, individual nucleotides do not contribute independently of the absolute strand break cross section of the whole DNA strand. The absolute quantification of sequence specific strand breaks will help develop a more accurate molecular level understanding of radiation induced DNA damage, which can then be used for optimized risk estimates in cancer radiation therapy.
Topics: DNA; DNA Damage; Electrons; Models, Chemical; Nanostructures; Oligonucleotides; Poly A
PubMed: 31877939
DOI: 10.3390/ijms21010111 -
ELife Aug 2020Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in...
Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3' mRNA isoforms for most genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.
Topics: Poly A; Polyadenylation; RNA Polymerase II; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Elongation, Genetic
PubMed: 32845240
DOI: 10.7554/eLife.59810 -
Molecular and Cellular Biology Sep 2022The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated...
The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated regions (3' UTRs) but rarely within coding regions. An individual gene can yield many 3'-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3' UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3' UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3'-UTR chimeric genes indicate that the poly(A) profile is determined by the 3' UTR. Thus, 3' UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3'-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3' UTRs.
Topics: 3' Untranslated Regions; 5' Untranslated Regions; Poly A; Polyadenylation; Protein Isoforms; RNA Isoforms; RNA, Messenger; Saccharomyces cerevisiae
PubMed: 35972270
DOI: 10.1128/mcb.00244-22 -
RNA (New York, N.Y.) Jul 2001The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of...
The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3' splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5' pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an approximately 62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.
Topics: Base Sequence; Exons; HeLa Cells; Humans; Introns; Molecular Sequence Data; Nuclear Proteins; Poly A; RNA Processing, Post-Transcriptional
PubMed: 11453064
DOI: 10.1017/s1355838201010329