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Molecular Cell Jun 2024Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the...
Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges.
Topics: Eukaryotic Initiation Factor-4E; Saccharomyces cerevisiae Proteins; Protein Biosynthesis; Amino Acids; Saccharomyces cerevisiae; Gene Expression Regulation, Fungal; RNA, Messenger; 5' Untranslated Regions; Basic-Leucine Zipper Transcription Factors; Cyclins; Poly(A)-Binding Proteins
PubMed: 38848691
DOI: 10.1016/j.molcel.2024.05.008 -
Molecular Cell Jun 2024Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive....
Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.
Topics: RNA, Circular; Neurons; Active Transport, Cell Nucleus; Adenosine; Cell Nucleus; Neurogenesis; Humans; Poly(A)-Binding Protein I; Animals; RNA; Cell Line; Cell Differentiation; Cytoplasm; Prosencephalon
PubMed: 38838666
DOI: 10.1016/j.molcel.2024.05.011 -
RNA Biology Jan 2024Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics....
Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.
Topics: RNA, Messenger; Polyribonucleotide Nucleotidyltransferase; Humans; Oligonucleotides; RNA Stability
PubMed: 38836544
DOI: 10.1080/15476286.2024.2363029 -
Frontiers in Microbiology 2024Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium as an obligate...
Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80-90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from containing the symbiont (Bm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5'-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of mRNA reads, resulting in a 16-1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the depletion probes can remove rRNA from the filarial worm and the majority of rRNA from the more distantly related free living nematode . These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.
PubMed: 38832111
DOI: 10.3389/fmicb.2024.1418032 -
Bioelectrochemistry (Amsterdam,... Oct 2024Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel...
Triblock polyadenine-based electrochemical aptasensor for ultra-sensitive detection of carcinoembryonic antigen via exonuclease III-assisted target recycling and hybridization chain reaction.
Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel electrochemical aptasensor using a triblock polyadenine probe for ultra-sensitive detection of CEA. The method leverages Exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction. The triblock polyadenine probe self-assembles on the bare gold electrode through the strong affinity between adenine and gold electrode, blocking CEA diffusion and providing a large immobilization surface. CEA binding to hairpin probe 1 (HP1), followed by the hybridization between HP1 and hairpin probe 2 (HP2), triggers DNA cleavage by Exo III, amplifying the signal via a hybridization chain reaction and producing numerous dsDNA walkers that generates a dramatic electrochemical impedance signal. Under optimized conditions, the aptasensor achieved two ultra-low detection limits: 0.39 ag∙mL within the concentration range of 5 ag∙mL to 5 × 10 ag∙mL, and 1.5 ag∙mL within the concentration range of 5 × 10 ag∙mL to 1 × 10 ag∙mL. Its performance in human serum samples meets the practical standards, offering a promising new tool for ultrasensitive tumor marker detection, potentially revolutionizing early cancer diagnosis.
Topics: Exodeoxyribonucleases; Carcinoembryonic Antigen; Humans; Nucleic Acid Hybridization; Biosensing Techniques; Electrochemical Techniques; Aptamers, Nucleotide; Limit of Detection; Poly A; Gold; Electrodes
PubMed: 38823375
DOI: 10.1016/j.bioelechem.2024.108749 -
Reproduction (Cambridge, England) Aug 2024Ovarian aging results in reactive oxygen species accumulation and mitochondrial deterioration. During the aging process, GRSF1 deficiency attenuates mitochondrial...
IN BRIEF
Ovarian aging results in reactive oxygen species accumulation and mitochondrial deterioration. During the aging process, GRSF1 deficiency attenuates mitochondrial function in aging granulosa cells.
ABSTRACT
Ovarian aging critically influences reproductive potential, with a marked decrease in oocyte quality and quantity and an increase in oxidative stress and mitochondrial dysfunction. This study elucidates the role of guanine-rich RNA sequence binding factor 1 (GRSF1) in the aging of ovarian granulosa cells (GCs). We observed a significant reduction in GRSF1 within GCs correlating with patient age, utilizing clinical samples from IVF patients. Using an siRNA-mediated knockdown technique, we established that diminished GRSF1 expression exacerbates mitochondrial dysfunction, elevates reactive oxygen species, and impairs ATP production. Furthermore, RNA immunoprecipitation revealed GRSF1's interaction with superoxide dismutase 2 (SOD2) mRNA, a key antioxidant enzyme, suggesting a mechanism whereby GRSF1 modulates oxidative stress. Downregulation of SOD2 reversed the protective effects of GRSF1 overexpression on mitochondrial function. These insights into the role of GRSF1 in ovarian aging may guide the development of interventions to improve fertility outcomes in advanced age.
Topics: Female; Granulosa Cells; Humans; Mitochondria; Reactive Oxygen Species; Oxidative Stress; Aging; Cellular Senescence; Adult; Superoxide Dismutase; Cells, Cultured; Poly(A)-Binding Proteins
PubMed: 38819377
DOI: 10.1530/REP-24-0015 -
Chembiochem : a European Journal of... May 2024RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the...
RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.
PubMed: 38818670
DOI: 10.1002/cbic.202400202 -
Science China. Life Sciences Jun 2024Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized....
Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized. Here, with profiling of APA in colorectal cancer tissues and poly(A) signal editing, we firstly identified that the shortened 3' UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration. We found that liquid-liquid phase separation (LLPS) of PABPN1 is reduced albeit with higher expression in cancer, and the reduction of LLPS leads to the shortened 3' UTR of CTNNBIP1 and promotes cell proliferation and migration. Notably, the splicing factor SNRPD2 upregulated in colorectal cancer, can interact with glutamic-proline (EP) domain of PABPN1, and then disrupt LLPS of PABPN1, which attenuates the repression effect of PABPN1 on the proximal poly(A) sites. Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1, suggesting that regulation of APA by interfering LLPS of 3' end processing factor may have the potential as a new way for the treatment of cancer.
Topics: Humans; Colorectal Neoplasms; Cell Proliferation; Poly(A)-Binding Protein I; Cell Movement; Polyadenylation; 3' Untranslated Regions; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Phase Separation
PubMed: 38811444
DOI: 10.1007/s11427-023-2495-x -
BMC Genomics May 2024Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing...
BACKGROUND
Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied.
RESULTS
We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits.
CONCLUSIONS
As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.
Topics: Sequence Analysis, RNA; Nanopore Sequencing; Nanopores; Humans; Animals; RNA; High-Throughput Nucleotide Sequencing
PubMed: 38807060
DOI: 10.1186/s12864-024-10440-w -
Chemical Communications (Cambridge,... Jun 2024A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids...
A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids (polyA-SNA) to accurately regulate the densities of DNA tracks, the nanowalker showcases its capability to identify miRNA-21, miRNA-486, and miRNA-155 with quick kinetics and attomolar sensitivity, positioning it as a promising option for cancer clinical surveillance.
Topics: MicroRNAs; Humans; Nanostructures; Poly A; DNA; Stochastic Processes; Biosensing Techniques
PubMed: 38804211
DOI: 10.1039/d4cc01656d