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Life Science Alliance Aug 2024IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in...
IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing , we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.
Topics: Humans; Protein Biosynthesis; Transcription Factors; Stress, Physiological; DNA-Binding Proteins; K562 Cells; Activating Transcription Factor 4; Gene Deletion; Gene Expression Regulation; RNA, Transfer
PubMed: 38803225
DOI: 10.26508/lsa.202302554 -
PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.Viruses May 2024Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by...
Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.
Topics: Hepacivirus; Humans; Ribosomal Proteins; Internal Ribosome Entry Sites; RNA, Viral; Protein Biosynthesis; Virus Replication; RNA-Binding Proteins; Protein Binding; Hepatitis C; Host-Pathogen Interactions
PubMed: 38793620
DOI: 10.3390/v16050738 -
ELife May 2024Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a...
Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of . RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and β-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain () as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of -challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.
Topics: Animals; Candida albicans; Mice; Candidiasis; Fungal Vaccines; Disease Models, Animal; Virulence; Female; Cytokines; Biofilms
PubMed: 38787374
DOI: 10.7554/eLife.93760 -
Aging Cell May 2024The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted....
The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted. Nucleus pulposus cell (NPC) ferroptosis is one of the causes of IVDD. N6-methyladenosine (m6A) and its reader protein YTHDF1 regulate cellular activities by affecting RNA metabolism. However, the regulation of ferroptosis in NPCs by m6A-modified RNAs under hypoxic conditions has not been as well studied. In this study, through in vitro and in vivo experiments, we explored the underlying mechanism of HIF-1α and YTHDF1 in regulating ferroptosis in NPCs. The results indicated that the overexpression of HIF-1α or YTHDF1 suppressed NPC ferroptosis; conversely, the knockdown of HIF-1α or YTHDF1 increased ferroptosis levels in NPCs. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF-1α regulated YTHDF1 transcription by directly binding to its promoter region. Polysome profiling results showed that YTHDF1 promoted the translation of SLC7A11 and consequently the expression of the anti-ferroptosis protein GPX4 by binding to m6A-modified SLC7A11 mRNA. In conclusion, HIF-1α-induced YTHDF1 expression reduces NPC ferroptosis and delays IVDD by promoting SLC7A11 translation in a m6A-dependent manner.
PubMed: 38783692
DOI: 10.1111/acel.14210 -
NAR Cancer Jun 2024In this review, we explore the transformative impact of next generation sequencing technologies in the realm of translatomics (the study of how translational machinery... (Review)
Review
In this review, we explore the transformative impact of next generation sequencing technologies in the realm of translatomics (the study of how translational machinery acts on a genome-wide scale). Despite the expectation of a direct correlation between mRNA and protein content, the complex regulatory mechanisms that affect this relationship remark the limitations of standard RNA-seq approaches. Then, the review characterizes crucial techniques such as polysome profiling, ribo-seq, trap-seq, proximity-specific ribosome profiling, rnc-seq, tcp-seq, qti-seq and scRibo-seq. All these methods are summarized within the context of cancer research, shedding light on their applications in deciphering aberrant translation in cancer cells. In addition, we encompass databases and bioinformatic tools essential for researchers that want to address translatome analysis in the context of cancer biology.
PubMed: 38751936
DOI: 10.1093/narcan/zcae024 -
Research Square Apr 2024Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive...
BACKGROUND
Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive arthritis, and differences in neutrophil physiological responses such as a faster response to the chemorepellent SLIGKV. Little is known about the basis of sex-based differences in human neutrophils.
METHODS
Starting with human neutrophils from healthy donors, we used RNA-seq to examine total mRNA profiles, mRNAs not associated with ribosomes and thus not being translated, mRNAs in monosomes, and mRNAs in polysomes and thus heavily translated. We used mass spectrometry systems to identify proteins and phosphoproteins.
RESULTS
There were sex-based differences in the translation of 24 mRNAs. There were 132 proteins with higher levels in male neutrophils; these tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Male neutrophils had increased phosphorylation of 32 proteins. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils.
CONCLUSIONS
Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways, while female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.
PubMed: 38746380
DOI: 10.21203/rs.3.rs-4284171/v1 -
Biological Research May 2024Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to...
BACKGROUND
Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies.
RESULTS
Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo mice treated with sub-optimal and optimal ascorbate levels.
CONCLUSIONS
Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.
Topics: Animals; Ascorbic Acid; Liver; Female; Male; Proteomics; Mice; L-Gulonolactone Oxidase; Gene Expression Profiling; Transcriptome; Principal Component Analysis; Antioxidants
PubMed: 38735981
DOI: 10.1186/s40659-024-00509-x -
Communications Biology May 2024The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are...
The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.
Topics: Cryoelectron Microscopy; Capsid; Cell Extracts; Saccharomyces cerevisiae; RNA, Viral; RNA, Messenger
PubMed: 38730276
DOI: 10.1038/s42003-024-06204-7 -
Communications Biology Apr 2024Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome...
Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on protein mobility within the E. coli cytoplasm. We employ nanoscale single-molecule displacement mapping (SMdM) to determine the spatial distribution of the proteins and to meticulously track their diffusion. We show that the distribution of polysomes does not impact the lateral diffusion coefficients of proteins. However, the degradation of mRNA induced by rifampicin treatment leads to an increase in protein mobility within the cytoplasm. Additionally, we establish a significant correlation between cell aging, the asymmetric localization of protein aggregates and reduced diffusion coefficients at the cell poles. Notably, we observe variations in the hindrance of diffusion at the poles and the central nucleoid region for small and large proteins, and we reveal differences between the old and new pole of the cell. Collectively, our research highlights cellular processes and mechanisms responsible for spatially organizing the bacterial cytoplasm into domains with different structural features and apparent viscosity.
Topics: Escherichia coli; Cytoplasm; Escherichia coli Proteins; Diffusion
PubMed: 38678067
DOI: 10.1038/s42003-024-06216-3 -
Journal of Experimental & Clinical... Apr 2024Radiation therapy stands to be one of the primary approaches in the clinical treatment of malignant tumors. Nasopharyngeal Carcinoma, a malignancy predominantly treated...
BACKGROUND
Radiation therapy stands to be one of the primary approaches in the clinical treatment of malignant tumors. Nasopharyngeal Carcinoma, a malignancy predominantly treated with radiation therapy, provides an invaluable model for investigating the mechanisms underlying radiation therapy resistance in cancer. While some reports have suggested the involvement of circRNAs in modulating resistance to radiation therapy, the underpinning mechanisms remain unclear.
METHODS
RT-qPCR and in situ hybridization were used to detect the expression level of circCDYL2 in nasopharyngeal carcinoma tissue samples. The effect of circCDYL2 on radiotherapy resistance in nasopharyngeal carcinoma was demonstrated by in vitro and in vivo functional experiments. The HR-GFP reporter assay determined that circCDYL2 affected homologous recombination repair. RNA pull down, RIP, western blotting, IF, and polysome profiling assays were used to verify that circCDYL2 promoted the translation of RAD51 by binding to EIF3D protein.
RESULTS
We have identified circCDYL2 as highly expressed in nasopharyngeal carcinoma tissues, and it was closely associated with poor prognosis. In vitro and in vivo experiments demonstrate that circCDYL2 plays a pivotal role in promoting radiotherapy resistance in nasopharyngeal carcinoma. Our investigation unveils a specific mechanism by which circCDYL2, acting as a scaffold molecule, recruits eukaryotic translation initiation factor 3 subunit D protein (EIF3D) to the 5'-UTR of RAD51 mRNA, a crucial component of the DNA damage repair pathway to facilitate the initiation of RAD51 translation and enhance homologous recombination repair capability, and ultimately leads to radiotherapy resistance in nasopharyngeal carcinoma.
CONCLUSIONS
These findings establish a novel role of the circCDYL2/EIF3D/RAD51 axis in nasopharyngeal carcinoma radiotherapy resistance. Our work not only sheds light on the underlying molecular mechanism but also highlights the potential of circCDYL2 as a therapeutic sensitization target and a promising prognostic molecular marker for nasopharyngeal carcinoma.
Topics: Humans; Nasopharyngeal Carcinoma; Rad51 Recombinase; Mice; Animals; Radiation Tolerance; Recombinational DNA Repair; RNA, Circular; Nasopharyngeal Neoplasms; Cell Line, Tumor; Female; Male; Prognosis; Mice, Nude
PubMed: 38654320
DOI: 10.1186/s13046-024-03049-0