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Nature Communications Nov 2023The genomic distribution of cleavage and polyadenylation (polyA) sites should be co-evolutionally optimized with the local gene structure. Otherwise, spurious...
The genomic distribution of cleavage and polyadenylation (polyA) sites should be co-evolutionally optimized with the local gene structure. Otherwise, spurious polyadenylation can cause premature transcription termination and generate aberrant proteins. To obtain mechanistic insights into polyA site optimization across the human genome, we develop deep/machine learning models to identify genome-wide putative polyA sites at unprecedented nucleotide-level resolution and calculate their strength and usage in the genomic context. Our models quantitatively measure position-specific motif importance and their crosstalk in polyA site formation and cleavage heterogeneity. The intronic site expression is governed by the surrounding splicing landscape. The usage of alternative polyA sites in terminal exons is modulated by their relative locations and distance to downstream genes. Finally, we apply our models to reveal thousands of disease- and trait-associated genetic variants altering polyadenylation activity. Altogether, our models represent a valuable resource to dissect molecular mechanisms mediating genome-wide polyA site expression and characterize their functional roles in human diseases.
Topics: Humans; Polyadenylation; Nucleotides; Deep Learning; Transcription, Genetic; Genome, Human
PubMed: 37968271
DOI: 10.1038/s41467-023-43266-3 -
Development (Cambridge, England) Jun 2022As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology....
As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoprotein (RNP) granules for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNP granules to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a previously unreported mechanism underlying uncoupled translation and transcription in haploid male mouse germ cells.
Topics: Animals; Haploidy; Male; Mice; MicroRNAs; Poly A; Protein Biosynthesis; RNA, Messenger; Spermatids
PubMed: 35588208
DOI: 10.1242/dev.199573 -
Genome Biology Oct 2018In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes...
BACKGROUND
In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration.
RESULTS
RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3' ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration.
CONCLUSIONS
Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration.
Topics: Cell Cycle; Cell Movement; Cells, Cultured; Fibroblasts; Humans; Poly A; Polyadenylation; RNA Splicing; Skin; mRNA Cleavage and Polyadenylation Factors
PubMed: 30360761
DOI: 10.1186/s13059-018-1551-9 -
International Journal of Molecular... Apr 2023Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist...
Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.
Topics: Humans; Polyadenylation; Cartilage, Articular; Transcriptome; Osteoarthritis, Knee; Sequence Analysis, RNA; RNA
PubMed: 37047586
DOI: 10.3390/ijms24076611 -
Molecular Biology and Evolution Jun 2023Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for...
Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3'-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in nonanimal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, and cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs, and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.
Topics: Animals; Phylogeny; Polyadenylation; Ctenophora; Sea Anemones
PubMed: 37288606
DOI: 10.1093/molbev/msad137 -
Wiley Interdisciplinary Reviews. RNA Jul 2019Alternative polyadenylation (APA) is how genes choose different sites for 3' end formation for mRNAs during transcription. APA often occurs in a tissue- or developmental... (Review)
Review
Alternative polyadenylation (APA) is how genes choose different sites for 3' end formation for mRNAs during transcription. APA often occurs in a tissue- or developmental stage-specific manner that can significantly affect gene activity by changing the protein product generated, the stability of the transcript, its localization within the cell, or its translatability. Despite the important regulatory effects that APA has on tissue-specific gene expression, only a few examples have been characterized mechanistically. In this 2018 update to our 2010 review, we examine mechanisms for the control of APA and update our understanding of the older mechanisms since 2010. We once postulated the existence of tissue-specific factors in APA. However, while a few tissue-specific polyadenylation factors are known, the emerging conclusion is that the majority of APA is accomplished by altering levels of core polyadenylation proteins. Examples of those core proteins include CSTF2, CPSF1, and subunits of mammalian cleavage factor I. But despite support for these mechanisms, no one has yet documented any of these proteins changing in either a tissue-specific or developmental manner. Given the profound effect that APA can have on gene expression and human health, improved understanding of tissue-specific APA could lead to numerous advances in gene activity control. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Development.
Topics: Brain; Gene Expression Regulation; Humans; Male; Polyadenylation; RNA, Messenger; RNA-Binding Proteins; Testis
PubMed: 30816016
DOI: 10.1002/wrna.1526 -
Cell Jun 2019Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of...
Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
Topics: HEK293 Cells; Humans; Poly A; Protein Domains; RNA Polymerase II; RNA, Messenger; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Transcription Elongation, Genetic; Transcription Termination, Genetic
PubMed: 31104839
DOI: 10.1016/j.cell.2019.04.038 -
BioEssays : News and Reviews in... Aug 2014Alternative cleavage and polyadenylation (APA) can diversify coding and non-coding regions, but has particular impact on increasing 3' UTR diversity. Through the gain or... (Review)
Review
Alternative cleavage and polyadenylation (APA) can diversify coding and non-coding regions, but has particular impact on increasing 3' UTR diversity. Through the gain or loss of regulatory elements such as RNA binding protein and microRNA sites, APA can influence transcript stability, localization, and translational efficiency. Strikingly, the central nervous systems of invertebrate and vertebrate species express a broad range of transcript isoforms bearing extended 3' UTRs. The molecular mechanism that permits proximal 3' end bypass in neurons is mysterious, and only beginning to be elucidated. This landscape of neural 3' UTR extensions, many reaching unprecedented lengths, may help service the unique post-transcriptional regulatory needs of neurons. A combination of approaches, including transcriptome-wide profiling, genetic screening to identify APA factors, biochemical dissection of alternative 3' end formation, and manipulation of individual neural APA targets, will be necessary to gain fuller perspectives on the mechanism and biology of neural-specific 3' UTR lengthening.
Topics: 3' Untranslated Regions; Animals; Central Nervous System; Gene Expression Regulation; Humans; Molecular Sequence Annotation; Organ Specificity; Polyadenylation; RNA Stability; RNA Transport
PubMed: 24903459
DOI: 10.1002/bies.201300174 -
Nature Communications Jan 2017Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate...
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
Topics: Genetic Techniques; Mutation; Poly A; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; RNA Stability
PubMed: 28106166
DOI: 10.1038/ncomms14112 -
Genes Nov 2023Manipulation using alternative exon splicing (AES), alternative transcription start (ATS), and alternative polyadenylation (APA) sites are key to transcript diversity... (Review)
Review
Manipulation using alternative exon splicing (AES), alternative transcription start (ATS), and alternative polyadenylation (APA) sites are key to transcript diversity underlying health and disease. All three are pervasive in organisms, present in at least 50% of human protein-coding genes. In fact, ATS and APA site use has the highest impact on protein identity, with their ability to alter which first and last exons are utilized as well as impacting stability and translation efficiency. These RNA variants have been shown to be highly specific, both in tissue type and stage, with demonstrated importance to cell proliferation, differentiation and the transition from fetal to adult cells. While alternative exon splicing has a limited effect on protein identity, its ubiquity highlights the importance of these minor alterations, which can alter other features such as localization. The three processes are also highly interwoven, with overlapping, complementary, and competing factors, RNA polymerase II and its CTD (C-terminal domain) chief among them. Their role in development means dysregulation leads to a wide variety of disorders and cancers, with some forms of disease disproportionately affected by specific mechanisms (AES, ATS, or APA). Challenges associated with the genome-wide profiling of RNA variants and their potential solutions are also discussed in this review.
Topics: Humans; RNA, Messenger; Polyadenylation; Alternative Splicing; Exons; Cell Differentiation
PubMed: 38002994
DOI: 10.3390/genes14112051