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The Journal of Biological Chemistry Jul 2023Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature...
Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.
Topics: Morpholinos; Oligonucleotides, Antisense; Polyadenylation; Ribonucleoprotein, U1 Small Nuclear; RNA Polymerase II; RNA Precursors; Humans; HeLa Cells; Gene Knockdown Techniques; Cleavage And Polyadenylation Specificity Factor; Cleavage Stimulation Factor; Transcription, Genetic
PubMed: 37224962
DOI: 10.1016/j.jbc.2023.104854 -
Cell Reports Apr 2023Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is...
Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.
Topics: RNA, Long Noncoding; Polyadenylation; RNA, Messenger; Telomere
PubMed: 37060569
DOI: 10.1016/j.celrep.2023.112406 -
Molecular Psychiatry Jul 2023The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic... (Review)
Review
The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic plasticity, the underlying cellular basis of learning and memory [1, 2]. The newly made proteins were proposed to "tag" the stimulated synapse, distinguishing it from naive synapses, thereby forming a cellular memory [3]. Subsequent studies demonstrated that the transport of mRNAs from soma to dendrite was linked with translational unmasking at synapses upon synaptic stimulation. It soon became apparent that one prevalent mechanism governing these events is cytoplasmic polyadenylation, and that among the proteins that control this process, CPEB, plays a central role in synaptic plasticity, and learning and memory. In vertebrates, CPEB is a family of four proteins, all of which regulate translation in the brain, that have partially overlapping functions, but also have unique characteristics and RNA binding properties that make them control different aspects of higher cognitive function. Biochemical analysis of the vertebrate CPEBs demonstrate them to respond to different signaling pathways whose output leads to specific cellular responses. In addition, the different CPEBs, when their functions go awry, result in pathophysiological phenotypes resembling specific human neurological disorders. In this essay, we review key aspects of the vertebrate CPEB proteins and cytoplasmic polyadenylation within the context of brain function.
Topics: Animals; Humans; Polyadenylation; Transcription Factors; mRNA Cleavage and Polyadenylation Factors; Protein Biosynthesis; Neuronal Plasticity
PubMed: 37131078
DOI: 10.1038/s41380-023-02088-x -
Wiley Interdisciplinary Reviews. RNA 2023Evolution and change generated an incredible diversity of organisms on this earth. Yet, some processes are so central to life that change is strongly selected against.... (Review)
Review
Evolution and change generated an incredible diversity of organisms on this earth. Yet, some processes are so central to life that change is strongly selected against. Synthesis of the eukaryotic messenger RNA is one example. The assemblies that carry out transcription and processing (capping, polyadenylation, and splicing) are so conserved that most genes have recognizable orthologs in yeast and humans. Naturally, most would conclude transcription and processing are identical in both sexes. However, this is an assumption. Men and women vastly differ in their physiologies. The incidence of pathologies, symptom presentation, disease outcome, and therapeutic response in each sex vary enormously. Despite the harm ignorance causes women, biological research has been historically carried out without regard to sex. The male mouse was the default mammal. A cultured cell's sex was considered irrelevant. Attempts to fill this knowledge gap have revealed molecular dissimilarities. For example, the earliest embryonic male and female transcriptomes differ long before fetal sex hormones appear. We used public data to challenge the assumption of sameness by reviewing reports of sex-biased gene expression and gene targeting. We focused on 120 genes encoding nonregulatory proteins involved in mRNA synthesis. Remarkably, genes with recognizable orthologs in yeast and thus LEAST likely to differ, did differ between the sexes. The rapidly growing public databases can be used to compare the expression of any gene in male and female tissues. Appreciating the principles that drive sex differences will enrich our understanding of RNA biology in all humans-men and women. This article is categorized under: RNA in Disease and Development > RNA in Development RNA Evolution and Genomics > Computational Analyses of RNA.
Topics: Female; Male; Humans; Animals; Mice; Saccharomyces cerevisiae; Transcription, Genetic; RNA Splicing; Polyadenylation; RNA, Messenger; Mammals
PubMed: 36195437
DOI: 10.1002/wrna.1765 -
Trends in Cell Biology Jan 2020Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative... (Review)
Review
Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative splicing and alternative polyadenylation result in greater transcript and protein diversity as well as function and activity. A critical role for alternative pre-mRNA processing in cell migration has emerged in axon outgrowth during neuronal development, immune cell migration, and cancer metastasis. These findings suggest that migratory signals result in expression changes of post-translational modifications of splicing or polyadenylation factors, leading to splicing events that generate promigratory isoforms. We summarize this recent progress and suggest emerging technologies that may facilitate a deeper understanding of the role of alternative splicing and polyadenylation in cell migration.
Topics: Alternative Splicing; Animals; Cell Movement; Humans; Models, Biological; Polyadenylation; Protein Isoforms; RNA Processing, Post-Transcriptional
PubMed: 31810769
DOI: 10.1016/j.tcb.2019.10.007 -
Cells Aug 2020RNA-binding proteins control the metabolism of RNAs at all stages of their lifetime. They are critically required for the post-transcriptional regulation of gene... (Review)
Review
RNA-binding proteins control the metabolism of RNAs at all stages of their lifetime. They are critically required for the post-transcriptional regulation of gene expression in a wide variety of physiological and pathological processes. Rbm24 is a highly conserved RNA-binding protein that displays strongly regionalized expression patterns and exhibits dynamic changes in subcellular localization during early development. There is increasing evidence that it acts as a multifunctional regulator to switch cell fate determination and to maintain tissue homeostasis. Dysfunction of Rbm24 disrupts cell differentiation in nearly every tissue where it is expressed, such as skeletal and cardiac muscles, and different head sensory organs, but the molecular events that are affected may vary in a tissue-specific, or even a stage-specific manner. Recent works using different animal models have uncovered multiple post-transcriptional regulatory mechanisms by which Rbm24 functions in key developmental processes. In particular, it represents a major splicing factor in muscle cell development, and plays an essential role in cytoplasmic polyadenylation during lens fiber cell terminal differentiation. Here we review the advances in understanding the implication of Rbm24 during development and disease, by focusing on its regulatory roles in physiological and pathological conditions.
Topics: Alternative Splicing; Animals; Cell Differentiation; Cell Lineage; Cytoplasm; Embryonic Development; Gene Expression Regulation; Homeostasis; Humans; Muscle Cells; Polyadenylation; RNA-Binding Proteins
PubMed: 32806768
DOI: 10.3390/cells9081891 -
Cells Feb 2022Repeat expansion diseases are a group of more than 40 disorders that affect mainly the nervous and/or muscular system and include myotonic dystrophies, Huntington's... (Review)
Review
Repeat expansion diseases are a group of more than 40 disorders that affect mainly the nervous and/or muscular system and include myotonic dystrophies, Huntington's disease, and fragile X syndrome. The mutation-driven expanded repeat tract occurs in specific genes and is composed of tri- to dodeca-nucleotide-long units. Mutant mRNA is a pathogenic factor or important contributor to the disease and has great potential as a therapeutic target. Although repeat expansion diseases are quite well known, there are limited studies concerning polyadenylation events for implicated transcripts that could have profound effects on transcript stability, localization, and translation efficiency. In this review, we briefly present polyadenylation and alternative polyadenylation (APA) mechanisms and discuss their role in the pathogenesis of selected diseases. We also discuss several methods for poly(A) tail measurement (both transcript-specific and transcriptome-wide analyses) and APA site identification-the further development and use of which may contribute to a better understanding of the correlation between APA events and repeat expansion diseases. Finally, we point out some future perspectives on the research into repeat expansion diseases, as well as APA studies.
Topics: Fragile X Syndrome; Humans; Huntington Disease; Polyadenylation; RNA, Messenger
PubMed: 35203324
DOI: 10.3390/cells11040677 -
Cardiovascular Research Jun 2023Cleavage and polyadenylation of pre-mRNAs is a necessary step for gene expression and function. Majority of human genes exhibit multiple polyadenylation sites, which can... (Review)
Review
Cleavage and polyadenylation of pre-mRNAs is a necessary step for gene expression and function. Majority of human genes exhibit multiple polyadenylation sites, which can be alternatively used to generate different mRNA isoforms from a single gene. Alternative polyadenylation (APA) of pre-mRNAs is important for the proteome and transcriptome landscape. APA is tightly regulated during development and contributes to tissue-specific gene regulation. Mis-regulation of APA is linked to a wide range of pathological conditions. APA-mediated gene regulation in the heart is emerging as a new area of research. Here, we will discuss the impact of APA on gene regulation during heart development and in cardiovascular diseases. First, we will briefly review how APA impacts gene regulation and discuss molecular mechanisms that control APA. Then, we will address APA regulation during heart development and its dysregulation in cardiovascular diseases. Finally, we will discuss pre-mRNA targeting strategies to correct aberrant APA patterns of essential genes for the treatment or prevention of cardiovascular diseases. The RNA field is blooming due to advancements in RNA-based technologies. RNA-based vaccines and therapies are becoming the new line of effective and safe approaches for the treatment and prevention of human diseases. Overall, this review will be influential for understanding gene regulation at the RNA level via APA in the heart and will help design RNA-based tools for the treatment of cardiovascular diseases in the future.
Topics: Humans; Polyadenylation; Cardiovascular Diseases; RNA, Messenger; Gene Expression Regulation; 3' Untranslated Regions; Heart
PubMed: 36657944
DOI: 10.1093/cvr/cvad014 -
Cancer Letters Sep 2019Alternative polyadenylation (APA) is now widely recognized to regulate gene expression. APA is an RNA-processing mechanism that generates distinct 3' termini on mRNAs,... (Review)
Review
Alternative polyadenylation (APA) is now widely recognized to regulate gene expression. APA is an RNA-processing mechanism that generates distinct 3' termini on mRNAs, producing mRNA isoforms. Different factors influence the initiation and development of this process. CFIm25 (among others) is a cleavage and polyadenylation factor that plays a key role in the regulation of APA. Shortening of the 3'UTRs on mRNAs leads to enhanced cellular proliferation and tumorigenicity. One reason may be the up-regulation of growth promoting factors, such as Cyclin D1. Different studies have reported a dual role of CFIm25 in cancer (both oncogenic and tumor suppressor). microRNAs (miRNAs) may be involved in CFIm25 function as well as competing endogenous RNAs (ceRNAs). The present review focuses on the role of CFIm25 in cancer, cancer treatment, and possible involvement in other human diseases. We highlight the involvement of miRNAs and ceRNAs in the function of CFIm25 to affect gene expression. The lack of understanding of the mechanisms and regulation of CFIm25 and APA has underscored the need for further research regarding their role in cancer and other diseases.
Topics: Animals; Cell Line, Tumor; Cleavage And Polyadenylation Specificity Factor; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Polyadenylation
PubMed: 31181319
DOI: 10.1016/j.canlet.2019.114430 -
Frontiers in Immunology 2023Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR...
INTRODUCTION
Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3'UTR-APA and IPA in primary human macrophages was unclear.
METHODS
In this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3'RNA-Seq was performed to quantify gene expression and characterize new 3'UTR-APA and IPA mRNA isoforms.
RESULTS
Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3'UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3'UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, is the pre-mRNA processing gene that is most upregulated. After knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses.
DISCUSSION
Our results reveal new 3'UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for in pro-inflammatory macrophages, key cells in the tumor response.
Topics: Humans; Polyadenylation; 3' Untranslated Regions; RNA Isoforms; Macrophages; Colorectal Neoplasms; Tumor Microenvironment
PubMed: 37359548
DOI: 10.3389/fimmu.2023.1182525