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Molecular Reproduction and Development Nov 2019Embryonic poly(A)-binding protein (EPAB) is an RNA-binding protein that binds to the poly(A) tails and AU-rich element at the 3' ends of messenger RNA (mRNAs). The main... (Review)
Review
Embryonic poly(A)-binding protein (EPAB) is an RNA-binding protein that binds to the poly(A) tails and AU-rich element at the 3' ends of messenger RNA (mRNAs). The main functions of EPAB are to protect stored mRNAs from undergoing deadenylation and subsequent degradation and to be involved in their translational regulation during spermatogenesis, oogenesis, and early embryogenesis. Following the first characterization of Epab in the Xenopus oocytes and early embryos, spatial and temporal expression and potential roles of the Epab gene have been determined in the vertebrate germ cells and early embryos. In this review, we have comprehensively evaluated all studies in this field and discussed the particular functions of EPAB in the spermatogenic cells, oocytes, early embryos, and somatic cells in vertebrates.
Topics: 3' Untranslated Regions; Animals; Embryo, Nonmammalian; Embryonic Development; Female; Male; Oogenesis; Poly(A)-Binding Proteins; Spermatogenesis; Xenopus Proteins; Xenopus laevis
PubMed: 31408254
DOI: 10.1002/mrd.23253 -
ELife Nov 2022Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that...
Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.
Topics: Humans; Polyadenylation; Nucleotides; RNA Polymerase II; Poly A; Saccharomyces cerevisiae; 3' Untranslated Regions; Transcription, Genetic
PubMed: 36421680
DOI: 10.7554/eLife.83153 -
Journal of Experimental & Clinical... Feb 2021Occurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3'ends, thus greatly expanding the diversity of... (Review)
Review
Occurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3'ends, thus greatly expanding the diversity of mRNAs and of proteins derived from a single gene. As a key molecular mechanism, APA is involved in various gene regulation steps including mRNA maturation, mRNA stability, cellular RNA decay, and protein diversification. APA is frequently dysregulated in cancers leading to changes in oncogenes and tumor suppressor gene expressions. Recent studies have revealed various APA regulatory mechanisms that promote the development and progression of a number of human diseases, including cancer. Here, we provide an overview of four types of APA and their impacts on gene regulation. We focus particularly on the interaction of APA with microRNAs, RNA binding proteins and other related factors, the core pre-mRNA 3'end processing complex, and 3'UTR length change. We also describe next-generation sequencing methods and computational tools for use in poly(A) signal detection and APA repositories and databases. Finally, we summarize the current understanding of APA in cancer and provide our vision for future APA related research.
Topics: 3' Untranslated Regions; Animals; Computational Biology; Databases, Genetic; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Poly A; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; RNA-Binding Proteins; Signal Transduction
PubMed: 33526057
DOI: 10.1186/s13046-021-01852-7 -
Molecular Therapy. Nucleic Acids Dec 2021Manipulation of gene activity through creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that...
Manipulation of gene activity through creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created by inserting -regulatory sequences composed of consecutive adenosine nucleotides called poly(A) tracks. Here we use poly(A) tracks to create hypomorphic mutants and functional characterization of membrane, secretory, and endogenous proteins. Insertion of poly(A) tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of a signaling sequence. Likewise, CRISPR-Cas9 targeted insertion of poly(A) tracks into the coding sequence of the endogenous human genes and results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1-engineered hypomorphs indicate a direct correlation between gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect expression of the target genes differentially depending on the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in poly(A) track-engineered cells as in cancer cells, indicating these variants' biological relevance. These results highlight this technology's power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR-Cas9 engineering tools.
PubMed: 34729253
DOI: 10.1016/j.omtn.2021.10.005 -
Chemical Communications (Cambridge,... Mar 2022Small interfering RNA (siRNA)-based therapeutics represent a novel and compelling drug modality, provided that safe and competent vectors are available for their...
Small interfering RNA (siRNA)-based therapeutics represent a novel and compelling drug modality, provided that safe and competent vectors are available for their delivery. Here, we report a biodegradable amphiphilic poly(aminoester) dendrimer for effective siRNA delivery. This dendrimer is readily biodegradable upon enzyme action, and harnesses the delivery features of both lipid and polymer vectors thanks to its lipid/dendrimer hybrid structure. This study opens new perspectives for developing biodegradable and biocompatible vectors for siRNA therapeutics.
Topics: Dendrimers; Poly A; RNA, Small Interfering
PubMed: 35094034
DOI: 10.1039/d1cc06655b -
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 -
RNA (New York, N.Y.) Sep 2020Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic...
Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic conditions are held together by A-A base-pairing also involving base interactions with the phosphate backbone. Traditional UV-melting studies of parallel poly(A) duplexes have typically examined homo-duplex formation of a single nucleic acid species in solution. We have adapted a technique utilizing a DNA nanoswitch that detects interaction of two different strands either with similar or differing lengths or modifications. Our method detected parallel duplex formation as a function of length, chemical modifications, and pH, and at a sensitivity that required over 100-fold less concentration of sample than prior UV-melting methods. While parallel polyriboadenylic acid and poly-2'-O-methyl-adenylic acid homo-duplexes formed, we did not detect homo-duplexes of polydeoxyriboadenylic acid strands or poly-locked nucleic acid (LNA)-adenylic strands. Importantly however, a poly-locked nucleic acid (LNA)-adenylic strand, as well as a poly-2'-O-methyl-adenylic strand, formed a hetero-duplex with a polyriboadenylic strand. Overall, our work validates a new tool for studying parallel duplexes and reveals fundamental properties of poly(A) parallel duplex formation. Parallel duplexes may find use in DNA nanotechnology and in molecular biology applications such as a potential poly(rA) tail capture tool as an alternative to traditional oligo(dT) based purification.
Topics: Base Pairing; DNA; Nucleic Acid Conformation; Oligonucleotides; Poly A
PubMed: 32414856
DOI: 10.1261/rna.075408.120 -
Biochemistry. Biokhimiia Sep 2021"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a... (Review)
Review
"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.
Topics: Cryoelectron Microscopy; Eukaryota; Nucleic Acid Conformation; Poly A; Polyribosomes; RNA Caps; RNA, Messenger; Ribosome Subunits
PubMed: 34565311
DOI: 10.1134/S0006297921090030 -
Aging Cell Oct 2023Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear...
Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.
Topics: Humans; Muscular Dystrophy, Oculopharyngeal; Autophagosomes; Mutation; Poly(A)-Binding Protein I
PubMed: 37559347
DOI: 10.1111/acel.13949 -
ELife Jul 2021In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail...
In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.
Topics: Animals; Gene Expression Regulation; Poly A; Poly(A)-Binding Proteins; RNA, Messenger; Xenopus Proteins; Xenopus laevis
PubMed: 34213414
DOI: 10.7554/eLife.66493