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Biochemical Society Transactions Jun 2023The 3' untranslated region (3'UTR) of mRNA plays a key role in the post-transcriptional regulation of gene expression. Most eukaryotic protein-coding genes express 3'UTR... (Review)
Review
The 3' untranslated region (3'UTR) of mRNA plays a key role in the post-transcriptional regulation of gene expression. Most eukaryotic protein-coding genes express 3'UTR isoforms owing to alternative cleavage and polyadenylation (APA). The 3'UTR isoform expression profile of a cell changes in cell proliferation, differentiation, and stress conditions. Here, we review the emerging theme of regulation of 3'UTR isoforms in cell metabolic reprogramming, focusing on cell growth and autophagy responses through the mTOR pathway. We discuss regulatory events that converge on the Cleavage Factor I complex, a master regulator of APA in 3'UTRs, and recent understandings of isoform-specific m6A modification and endomembrane association in determining differential metabolic fates of 3'UTR isoforms.
Topics: 3' Untranslated Regions; RNA Isoforms; Gene Expression Regulation; Polyadenylation; Protein Isoforms
PubMed: 37171086
DOI: 10.1042/BST20221128 -
PLoS Pathogens Dec 2022"Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression... (Review)
Review
"Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There are several RNA base modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites-Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.
Topics: Animals; RNA; Parasites; Gene Expression Regulation; RNA Processing, Post-Transcriptional; Eukaryota; Polyadenylation
PubMed: 36548245
DOI: 10.1371/journal.ppat.1010972 -
International Journal of Molecular... May 2021Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to... (Review)
Review
Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.
Topics: 3' Untranslated Regions; Alternative Splicing; Biomarkers, Tumor; Computational Biology; Databases, Nucleic Acid; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Polyadenylation; RNA Splice Sites; RNA, Messenger; RNA-Seq; Single-Cell Analysis
PubMed: 34070203
DOI: 10.3390/ijms22105322 -
The Journal of Biological Chemistry Dec 2019Soluble extracts prepared from eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development,...
Soluble extracts prepared from eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts.
Topics: Animals; Cell Nucleus; Cell-Free System; Cytoplasm; Fertilization; Gene Expression Regulation; Genome; Histones; Micrococcal Nuclease; Oocytes; Plasmids; Polyadenylation; RNA Polymerase II; Sequence Analysis, RNA; Transcription Factors; Transcription, Genetic; Xenopus laevis
PubMed: 31732562
DOI: 10.1074/jbc.RA119.011350 -
Proceedings of the National Academy of... Dec 2023Postintegration transcriptional silencing of HIV-1 leads to the establishment of a pool of latently infected cells. In these cells, mechanisms controlling RNA Polymerase...
Postintegration transcriptional silencing of HIV-1 leads to the establishment of a pool of latently infected cells. In these cells, mechanisms controlling RNA Polymerase II (RNAPII) pausing and premature transcription termination (PTT) remain to be explored. Here, we found that the cleavage and polyadenylation (CPA) factor PCF11 represses HIV-1 expression independently of the other subunits of the CPA complex or the polyadenylation signal located at the 5' LTR. We show that PCF11 interacts with the RNAPII-binding protein WDR82. Knock-down of PCF11 or WDR82 reactivated HIV-1 expression in latently infected cells. To silence HIV-1 transcription, PCF11 and WDR82 are specifically recruited at the promoter-proximal region of the provirus in an interdependent manner. Codepletion of PCF11 and WDR82 indicated that they act on the same pathway to repress HIV expression. These findings reveal PCF11/WDR82 as a PTT complex silencing HIV-1 expression in latently infected cells.
Topics: HIV-1; Transcription, Genetic; RNA Polymerase II; mRNA Cleavage and Polyadenylation Factors; Polyadenylation; Virus Latency
PubMed: 38015843
DOI: 10.1073/pnas.2313356120 -
Developmental Cell Apr 2024During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated...
During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.
Topics: Animals; Oocytes; Polyadenylation; Protein Biosynthesis; Poly A; 3' Untranslated Regions; RNA, Messenger; Gene Expression Regulation, Developmental; Mice; Humans; Embryo, Nonmammalian; Embryonic Development; Female; Xenopus laevis; Cytoplasm
PubMed: 38460509
DOI: 10.1016/j.devcel.2024.02.007 -
Molecular Cell Oct 2022The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome....
The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.
Topics: Polyadenylation; RNA Isoforms; 5' Untranslated Regions; 3' Untranslated Regions; Proto-Oncogene Proteins c-bcl-2; Exonucleases
PubMed: 36270248
DOI: 10.1016/j.molcel.2022.09.036 -
Genes & Development Sep 2021Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its...
Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in , we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.
Topics: Polyadenylation; RNA, Messenger; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34385261
DOI: 10.1101/gad.348634.121 -
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 -
Biophysical Journal Feb 2022Nascent pre-mRNA 3'-end cleavage and polyadenylation (C/P) involves numerous proteins that recognize multiple RNA elements. Human CSTF2 binds to a downstream U- or...
Nascent pre-mRNA 3'-end cleavage and polyadenylation (C/P) involves numerous proteins that recognize multiple RNA elements. Human CSTF2 binds to a downstream U- or G/U-rich sequence through its RNA recognition motif (RRM) regulating C/P. We previously reported the only known disease-related CSTF2 RRM mutant (CSTF2) and showed that it changed the on-rate of RNA binding, leading to alternative polyadenylation in brains of mice carrying the same mutation. In this study, we further investigated the role of electrostatic interactions in the thermodynamics and kinetics of RNA binding for the CSTF2 RRM and the downstream consequences for regulation of C/P. By combining mutagenesis with NMR spectroscopy and biophysical assays, we confirmed that electrostatic attraction is the dominant factor in RRM binding to a naturally occurring U-rich RNA sequence. Moreover, we demonstrate that RNA binding is accompanied by an enthalpy-entropy compensation mechanism that is supported by changes in pico-to-nanosecond timescale RRM protein dynamics. We suggest that the dynamic binding of the RRM to U-rich RNA supports the diversity of sequences it encounters in the nucleus. Lastly, in vivo C/P assays demonstrate a competition between fast, high affinity RNA binding and efficient, correct C/P. These results highlight the importance of the surface charge of the RRM in RNA binding and the balance between nascent mRNA binding and C/P in vivo.
Topics: Animals; Mice; Polyadenylation; Protein Binding; RNA; RNA Precursors; RNA Recognition Motif; Static Electricity
PubMed: 35090899
DOI: 10.1016/j.bpj.2022.01.005