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Trends in Genetics : TIG Sep 2023Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated... (Review)
Review
Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.
Topics: RNA Precursors; Transcription, Genetic; RNA Splicing; Spliceosomes; Introns
PubMed: 37236814
DOI: 10.1016/j.tig.2023.04.008 -
Nature Mar 2024Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells, are formed by back-splicing of precursor mRNAs...
Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells, are formed by back-splicing of precursor mRNAs in the nucleus. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.
Topics: Active Transport, Cell Nucleus; Cell Nucleus; Guanosine Triphosphate; Karyopherins; Nuclear Proteins; ran GTP-Binding Protein; RNA, Circular; RNA Precursors; RNA-Binding Proteins; Exportin 1 Protein; RNA Transport; Protein Transport
PubMed: 38355801
DOI: 10.1038/s41586-024-07060-5 -
The Biochemical Journal Jan 2024RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA... (Review)
Review
RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine.
Topics: Pseudouridine; RNA Precursors; RNA, Guide, CRISPR-Cas Systems; RNA; RNA Processing, Post-Transcriptional; Protein Biosynthesis
PubMed: 38174858
DOI: 10.1042/BCJ20230096 -
Cell Research Jul 2023Genetic information is generally transferred from RNA to protein according to the classic "Central Dogma". Here, we made a striking discovery that post-translational...
Genetic information is generally transferred from RNA to protein according to the classic "Central Dogma". Here, we made a striking discovery that post-translational modification of a protein specifically regulates the editing of its own mRNA. We show that S-nitrosylation of cathepsin B (CTSB) exclusively alters the adenosine-to-inosine (A-to-I) editing of its own mRNA. Mechanistically, CTSB S-nitrosylation promotes the dephosphorylation and nuclear translocation of ADD1, leading to the recruitment of MATR3 and ADAR1 to CTSB mRNA. ADAR1-mediated A-to-I RNA editing enables the binding of HuR to CTSB mRNA, resulting in increased CTSB mRNA stability and subsequently higher steady-state levels of CTSB protein. Together, we uncovered a unique feedforward mechanism of protein expression regulation mediated by the ADD1/MATR3/ADAR1 regulatory axis. Our study demonstrates a novel reverse flow of information from the post-translational modification of a protein back to the post-transcriptional regulation of its own mRNA precursor. We coined this process as "Protein-directed EDiting of its Own mRNA by ADAR1 (PEDORA)" and suggest that this constitutes an additional layer of protein expression control. "PEDORA" could represent a currently hidden mechanism in eukaryotic gene expression regulation.
Topics: RNA Editing; RNA, Messenger; Cathepsin B; Gene Expression Regulation; RNA Precursors; RNA; Adenosine Deaminase
PubMed: 37156877
DOI: 10.1038/s41422-023-00812-4 -
Nucleic Acids Research Aug 2023The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that...
The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.
Topics: Animals; Humans; Mice; Alternative Splicing; Genomic Instability; Lung Neoplasms; R-Loop Structures; RNA Polymerase II; RNA, Messenger
PubMed: 37395406
DOI: 10.1093/nar/gkad548 -
Science (New York, N.Y.) Sep 2023Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long...
Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme.
Topics: Animals; Cryoelectron Microscopy; Histones; Polycomb Repressive Complex 2; RNA Precursors; RNA, Long Noncoding; Zebrafish; G-Quadruplexes; Gain of Function Mutation; Promoter Regions, Genetic; Protein Binding; Enhancer of Zeste Homolog 2 Protein; Crystallography, X-Ray; Protein Conformation; Protein Multimerization
PubMed: 37733873
DOI: 10.1126/science.adh0059 -
Molecular Cell Aug 2023Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice...
Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.
Topics: Humans; RNA Splice Sites; Introns; RNA Splicing Factors; RNA Splicing; RNA-Binding Proteins; RNA Precursors; DNA-Binding Proteins
PubMed: 37506698
DOI: 10.1016/j.molcel.2023.07.002 -
Brain, Behavior, and Immunity Jan 2024Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and...
BACKGROUND
Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aβ pathology, neuroinflammation, and brain lipids.
METHODS
For in vitro studies, U373 human astrocytoma cells were treated with Aβ fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFAD with PCSK9 mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aβ plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test.
RESULTS
In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aβ fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aβ burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels.
CONCLUSIONS
In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aβ pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.
Topics: Mice; Humans; Animals; Mice, Transgenic; Proprotein Convertase 9; Amyloid Precursor Protein Secretases; Neuroinflammatory Diseases; Chromatography, Liquid; Inflammasomes; Aspartic Acid Endopeptidases; Tandem Mass Spectrometry; Alzheimer Disease; Cognitive Dysfunction; RNA, Messenger; Cholesterol; Amyloid beta-Peptides; Disease Models, Animal
PubMed: 37967665
DOI: 10.1016/j.bbi.2023.11.008