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Cancers Apr 2024FAM46C is a well-established tumour suppressor with a role that is not completely defined or universally accepted. Although FAM46C expression is down-modulated in... (Review)
Review
FAM46C is a well-established tumour suppressor with a role that is not completely defined or universally accepted. Although FAM46C expression is down-modulated in several tumours, significant mutations in the gene are only found in multiple myeloma (MM). Consequently, its tumour suppressor activity has primarily been studied in the MM context. However, emerging evidence suggests that FAM46C is involved also in other cancer types, namely colorectal, prostate and gastric cancer and squamous cell and hepatocellular carcinoma, where FAM46C expression was found to be significantly reduced in tumoural versus non-tumoural tissues and where FAM46C was shown to possess anti-proliferative properties. Accordingly, FAM46C was recently proposed to function as a pan-cancer prognostic marker, bringing FAM46C under the spotlight and attracting growing interest from the scientific community in the pathways modulated by FAM46C and in its mechanistic activity. Here, we will provide the first comprehensive review regarding FAM46C by covering (1) the intracellular pathways regulated by FAM46C, namely the MAPK/ERK, PI3K/AKT, β-catenin and TGF-β/SMAD pathways; (2) the models regarding its mode of action, specifically the poly(A) polymerase, intracellular trafficking modulator and inhibitor of centriole duplication models, focusing on connections and interdependencies; (3) the regulation of FAM46C expression in different environments by interferons, IL-4, TLR engagement or transcriptional modulators; and, lastly, (4) how FAM46C expression levels associate with increased/decreased tumour cell sensitivity to anticancer agents, such as bortezomib, dexamethasone, lenalidomide, pomalidomide, doxorubicin, melphalan, SK1-I, docetaxel and norcantharidin.
PubMed: 38730656
DOI: 10.3390/cancers16091706 -
Biochemistry May 2024Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host...
Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host mRNAs and triggering their decay. Surprisingly, viral RNA, which resembles host mRNAs containing a 5'-cap and a 3'-poly(A) tail, escapes significant translation inhibition and RNA decay, aiding viral propagation. Current literature proposes that, in SCOV2, nsp1 binds the viral RNA leader sequence, and the interaction may serve to distinguish viral RNA from host mRNA. However, a direct binding between SCOV1 nsp1 and the corresponding RNA leader sequence has not been established yet. Here, we show that SCOV1 nsp1 binds to the SCOV1 RNA leader sequence but forms multiple complexes at a high concentration of nsp1. These complexes are marginally different from complexes formed with SCOV2 nsp1. Finally, mutations of the RNA stem-loop did not completely abolish RNA binding by nsp1, suggesting that an RNA secondary structure is more important for binding than the sequence itself. Understanding the nature of binding of nsp1 to viral RNA will allow us to understand how this viral protein selectively suppresses host gene expression.
Topics: RNA, Viral; Viral Nonstructural Proteins; Protein Binding; Humans; Severe acute respiratory syndrome-related coronavirus; 5' Untranslated Regions; SARS-CoV-2; RNA-Dependent RNA Polymerase
PubMed: 38718213
DOI: 10.1021/acs.biochem.4c00078 -
PloS One 2024The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection,...
The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation.
Topics: Phosphorylation; Potyvirus; Triticum; Protein Binding; Protein Biosynthesis; Eukaryotic Initiation Factors; Poly(A)-Binding Proteins; Plant Proteins; Viral Proteins; Casein Kinase II
PubMed: 38696388
DOI: 10.1371/journal.pone.0300287 -
PLoS Pathogens Apr 2024Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the...
Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.
Topics: Animals; Nidovirales; Genome, Viral; Coronavirus; Phylogeny; Vertebrates; Fishes; Evolution, Molecular; Data Mining; Nidovirales Infections
PubMed: 38648214
DOI: 10.1371/journal.ppat.1012163 -
Journal of Virology May 2024Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein...
Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.
Topics: Humans; A549 Cells; Host-Pathogen Interactions; Influenza A virus; Influenza, Human; RNA, Messenger; Viral Nonstructural Proteins; Virus Replication; Host-Parasite Interactions
PubMed: 38629840
DOI: 10.1128/jvi.01901-23 -
BioRxiv : the Preprint Server For... Apr 2024High-resolution annotations of transcriptomes from all domains of life are essential for many sequencing-based RNA analyses, including Nanopore direct RNA sequencing...
High-resolution annotations of transcriptomes from all domains of life are essential for many sequencing-based RNA analyses, including Nanopore direct RNA sequencing (DRS), which would otherwise be hindered by misalignments and other analysis artefacts. DRS allows the capture and full-length sequencing of native RNAs, without recoding or amplification bias, and resulting data may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails on individual RNA molecules. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene dense organisms such as viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS datasets and developed a novel approach to transcriptome. We demonstrate, using a combination of synthetic and original datasets, that our novel approach yields a high level of precision and recall when reconstructing both gene sparse and gene dense transcriptomes from DRS datasets. We further apply this approach to generate a new high resolution transcriptome annotation of the neglected pathogen human adenovirus type F 41 for which we identify 77 distinct transcripts encoding at least 23 different proteins.
PubMed: 38617228
DOI: 10.1101/2024.04.02.587744 -
Signal Transduction and Targeted Therapy Apr 2024
Topics: Humans; Ribonucleases; Neoplasms; MicroRNAs
PubMed: 38616203
DOI: 10.1038/s41392-024-01795-3 -
Nucleic Acids Research Jun 2024The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental...
The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.
Topics: Animals; Mice; Calcium-Binding Proteins; Cell Differentiation; DNA Methylation; Gene Expression Regulation, Developmental; Genomic Imprinting; Intercellular Signaling Peptides and Proteins; Membrane Proteins; MicroRNAs; Mouse Embryonic Stem Cells; Nuclear Proteins; Pregnancy Proteins; Promoter Regions, Genetic; RNA, Long Noncoding
PubMed: 38613389
DOI: 10.1093/nar/gkae247 -
Nature Communications Apr 2024The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with...
The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.
Topics: Humans; Spliceosomes; RNA Splice Sites; Proteomics; RNA Splicing; Alternative Splicing; RNA, Small Nuclear; RNA, Messenger; Mutation; DNA Helicases; RNA-Binding Proteins; Retinitis Pigmentosa
PubMed: 38605034
DOI: 10.1038/s41467-024-47253-0 -
Nature Communications Apr 2024Cells must sense and respond to sudden maladaptive environmental changes-stresses-to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved...
Cells must sense and respond to sudden maladaptive environmental changes-stresses-to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide a remarkable view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein-a core marker of stress granules-condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry.
Topics: Heat-Shock Response; Stress, Physiological; Biological Evolution; Poly(A)-Binding Proteins
PubMed: 38605014
DOI: 10.1038/s41467-024-47355-9