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Nucleic Acids Research Nov 2023N 6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is...
N 6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is spatially regulated remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m6A sites in target RNAs which enables simultaneous detection of both m6A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m6A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m6A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m6A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m6A-modified transcript dynamics and investigating methylated RNA localization in individual cells.
Topics: RNA; RNA, Messenger; RNA Processing, Post-Transcriptional; In Situ Hybridization, Fluorescence
PubMed: 37811887
DOI: 10.1093/nar/gkad787 -
Nucleic Acids Research Dec 2023Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and...
Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.
Topics: Embryonic Development; Gene Expression Regulation, Developmental; Meiosis; Oocytes; Protein Biosynthesis; RNA, Messenger; Animals; Mice
PubMed: 37950888
DOI: 10.1093/nar/gkad996 -
CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.EMBO Reports Dec 2023Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host...
Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.
Topics: Humans; Transcription Factors; RNA, Messenger; Herpesviridae Infections; Receptors, CCR4
PubMed: 37846490
DOI: 10.15252/embr.202256327 -
Annual Review of Virology Sep 2023Protein synthesis by the ribosome is the final stage of biological information transfer and represents an irreversible commitment to gene expression. Accurate... (Review)
Review
Protein synthesis by the ribosome is the final stage of biological information transfer and represents an irreversible commitment to gene expression. Accurate translation of messenger RNA is therefore essential to all life, and spontaneous errors by the translational machinery are highly infrequent (∼1/100,000 codons). Programmed -1 ribosomal frameshifting (-1PRF) is a mechanism in which the elongating ribosome is induced at high frequency to slip backward by one nucleotide at a defined position and to continue translation in the new reading frame. This is exploited as a translational regulation strategy by hundreds of RNA viruses, which rely on -1PRF during genome translation to control the stoichiometry of viral proteins. While early investigations of -1PRF focused on virological and biochemical aspects, the application of X-ray crystallography and cryo-electron microscopy (cryo-EM), and the advent of deep sequencing and single-molecule approaches have revealed unexpected structural diversity and mechanistic complexity. Molecular players from several model systems have now been characterized in detail, both in isolation and, more recently, in the context of the elongating ribosome. Here we provide a summary of recent advances and discuss to what extent a general model for -1PRF remains a useful way of thinking.
Topics: Frameshifting, Ribosomal; Cryoelectron Microscopy; Ribosomes; RNA, Messenger; RNA Viruses; RNA, Viral
PubMed: 37339768
DOI: 10.1146/annurev-virology-111821-120646 -
Clinical and Translational Medicine Mar 2024NOP2/Sun domain 2 (NSUN2) is one of the important RNA methyltransferases catalyzing 5-methylcytosine (m5C) formation and participates in many critical bioprocesses....
BACKGROUND
NOP2/Sun domain 2 (NSUN2) is one of the important RNA methyltransferases catalyzing 5-methylcytosine (m5C) formation and participates in many critical bioprocesses. However, the roles and underlying molecular mechanisms of NSUN2-mediated m5C modification in colorectal cancer (CRC) remain unclear.
METHODS
To explore the NSUN2 expression in CRC, fresh tissue samples were collected and Nsun2 knockout mouse was constructed. In vitro and in vivo functional assays were conducted to assess the role of NSUN2. RNA array and bisulfite sequencing were used to investigate the potential targets. The mechanisms of NSUN2 function on SKIL were identified by m5C-methylated-RNA immunoprecipitation and RNA stability assays. Additionally, tissue microarray analysis was conducted and patient-derived tumour xenograft mouse (PDX) models were used to define the potential therapeutic targets.
RESULTS
NSUN2 was highly expressed in CRC and correlated with poor CRC patient survival. Moreover, silencing NSUN2 suppressed CRC tumourigenesis and progression in Nsun2 knockout mouse models. In vitro and in vivo studies suggested that NSUN2 promoted colorectal cancer cell growth. Mechanistically, SKI-like proto-oncogene (SKIL) is positively regulated by NSUN2, and the NSUN2-SKIL axis is clinically relevant to CRC. NSUN2 induced m5C modification of SKIL and stabilized its mRNA, which was mediated by Y-box binding protein 1 (YBX1). Elevated SKIL levels increased transcriptional coactivator with PDZ-binding motif (TAZ) activation.
CONCLUSIONS
Our findings highlight the importance of NSUN2 in the initiation and progression of CRC via m5C-YBX1-dependent stabilization of the SKIL transcript, providing a promising targeted therapeutic strategy for CRC.
Topics: Animals; Humans; Mice; Colorectal Neoplasms; Intracellular Signaling Peptides and Proteins; Methyltransferases; Mice, Knockout; Proto-Oncogene Proteins; RNA; RNA, Messenger
PubMed: 38468490
DOI: 10.1002/ctm2.1621 -
ELife Feb 2024A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.
A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.
Topics: Protein Biosynthesis; Ribosomes; RNA, Messenger
PubMed: 38393777
DOI: 10.7554/eLife.96304 -
Theranostics 2023Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) serve essential biological functions as post-transcriptional performers, participating in the acquisition or... (Review)
Review
Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) serve essential biological functions as post-transcriptional performers, participating in the acquisition or maintenance of tumor hallmarks due to their distinct protein structures. Emerging evidence indicates that IGF2BPs belong to the class III type of RNA N-methyladenosine (mA) modification readers, controlling RNA stability, storage, localization, metabolism, and translation in multiple vital bioprocesses, particularly tumorigenesis and tumor progression. Here, we discuss the underlying regulatory mechanisms and pathological functions of IGF2BPs which act as mA readers in the context of tumor pathogenesis and multidrug resistance. Furthermore, we highlight the potential of IGF2BPs as drug targets in clinical tumor treatment. Hence, precise and novel tumor therapeutic approaches could be uncovered by targeting epigenetic heterogeneity.
Topics: Humans; RNA, Messenger; Carrier Proteins; RNA-Binding Proteins; Neoplasms; RNA
PubMed: 37554271
DOI: 10.7150/thno.86528 -
Molecular Cell Aug 2023Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential...
Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.
Topics: RNA, Messenger; Oncogene Proteins; Cell Nucleus; Cytosol; Protein Transport; Protein Biosynthesis; Membrane Proteins
PubMed: 37506697
DOI: 10.1016/j.molcel.2023.06.036 -
Frontiers in Cellular and Infection... 2023RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted...
RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species.
Topics: Animals; RNA; Proteomics; RNA, Messenger; Extracellular Vesicles; RNA-Binding Proteins; Mammals
PubMed: 37780856
DOI: 10.3389/fcimb.2023.1247329 -
Genome Research Sep 2023Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their cytoarchitecture. The targeting of...
Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their cytoarchitecture. The targeting of RNA transcripts to axons is one of the mechanisms that allows rapid local translation of proteins in response to extracellular signals. 3' Untranslated regions (UTRs) of mRNA are noncoding sequences that play a critical role in determining transcript localization and translation by interacting with specific RNA-binding proteins (RBPs). However, how 3' UTRs contribute to mRNA metabolism and the nature of RBP complexes responsible for these functions remains elusive. We performed 3' end sequencing of RNA isolated from cell bodies and axons of sympathetic neurons exposed to either nerve growth factor (NGF) or neurotrophin 3 (NTF3, also known as NT-3). NGF and NTF3 are growth factors essential for sympathetic neuron development through distinct signaling mechanisms. Whereas NTF3 acts mostly locally, NGF signal is retrogradely transported from axons to cell bodies. We discovered that both NGF and NTF3 affect transcription and alternative polyadenylation in the nucleus and induce the localization of specific 3' UTR isoforms to axons, including short 3' UTR isoforms found exclusively in axons. The integration of our data with CLIP sequencing data supports a model whereby long 3' UTR isoforms associate with RBP complexes in the nucleus and, upon reaching the axons, are remodeled locally into shorter isoforms. Our findings shed new light into the complex relationship between nuclear polyadenylation, mRNA localization, and local 3' UTR remodeling in developing neurons.
Topics: Nerve Growth Factor; RNA, Messenger; 3' Untranslated Regions; Axons; Protein Isoforms; RNA-Binding Proteins
PubMed: 37582635
DOI: 10.1101/gr.277804.123