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International Journal of Biological... Jun 2024Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on...
Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94 cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188-750 cells/mL and 3000-6000 cells/mL (R ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.
Topics: Leishmania infantum; Chitosan; Nanocomposites; Aptamers, Nucleotide; Biosensing Techniques; Carbon; Limit of Detection; Fluorescent Dyes; Humans
PubMed: 38763252
DOI: 10.1016/j.ijbiomac.2024.132483 -
Molecular Systems Biology Jul 2024Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon...
Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.
Topics: Humans; Neurogenesis; Cell Differentiation; RNA Isoforms; Neurons; Alternative Splicing; RNA-Binding Proteins; Protein Isoforms; Exons
PubMed: 38755290
DOI: 10.1038/s44320-024-00039-4 -
PLoS Computational Biology May 2024The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked...
The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation.
Topics: RNA, Messenger; Humans; Cell Nucleus; Active Transport, Cell Nucleus; RNA Stability; 3' Untranslated Regions; Cell Line, Tumor; Cytosol
PubMed: 38753883
DOI: 10.1371/journal.pcbi.1012059 -
Nature Communications May 2024Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although...
Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although there exist over 1,500 RBPs in human cells, their binding motifs and functions still remain to be elucidated, especially in the complex environment of tissues and in the context of diseases. To overcome the lack of methods for the systematic and automated detection of sequence motif-guided pre-mRNA processing regulation from RNA sequencing (RNA-Seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). Applying MAPP to RBP knock-down experiments reveals that many RBPs regulate both splicing and polyadenylation of nascent transcripts by acting on similar sequence motifs. MAPP not only infers these sequence motifs, but also unravels the position-dependent impact of the RBPs on pre-mRNA processing. Interestingly, all investigated RBPs that act on both splicing and 3' end processing exhibit a consistently repressive or activating effect on both processes, providing a first glimpse on the underlying mechanism. Applying MAPP to normal and malignant brain tissue samples unveils that the motifs bound by the PTBP1 and RBFOX RBPs coordinately drive the oncogenic splicing program active in glioblastomas demonstrating that MAPP paves the way for characterizing pre-mRNA processing regulators under physiological and pathological conditions.
Topics: Humans; RNA-Binding Proteins; Polyadenylation; RNA Precursors; RNA Splicing; Gene Expression Regulation, Neoplastic; Neoplasms; Nucleotide Motifs; Polypyrimidine Tract-Binding Protein; RNA Splicing Factors; Heterogeneous-Nuclear Ribonucleoproteins; RNA, Messenger
PubMed: 38750024
DOI: 10.1038/s41467-024-48046-1 -
BioRxiv : the Preprint Server For... May 2024elements are non-autonomous Short INterspersed Elements (SINEs) derived from the gene that are present at over one million copies in human genomic DNA. mobilizes by a...
elements are non-autonomous Short INterspersed Elements (SINEs) derived from the gene that are present at over one million copies in human genomic DNA. mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) -encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support retrotransposition. Human elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 ( -permissive) strains, but not in HeLa-JVM or HeLa-H1 ( -nonpermissive) strains. A similar pattern of retrotransposition was observed for other -derived SINEs and -derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human - ( ) element, and an -containing messenger RNA can retrotranspose in all four HeLa strains. Using an reverse transcriptase-based assay, we show that RNAs associate with ORF2p and are converted into cDNAs in both -permissive and -nonpermissive HeLa strains, suggesting that - and -derived SINE RNAs use strategies to 'hijack' L1 ORF2p that are distinct from those used by elements and -containing mRNAs. These data further suggest ORF2p associates with the RNA poly(A) tract in both -permissive and -nonpermissive HeLa strains, but that retrotransposition is blocked after this critical step in -nonpermissive HeLa strains.
PubMed: 38746229
DOI: 10.1101/2024.05.03.592410 -
BMC Plant Biology May 2024Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore...
BACKGROUND
Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition.
RESULTS
A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions.
CONCLUSIONS
These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.
Topics: Transcriptome; Sequence Analysis, RNA; Nanopore Sequencing; Marchantia; Gene Expression Regulation, Plant; RNA, Plant; Adaptation, Physiological; Epigenesis, Genetic
PubMed: 38745128
DOI: 10.1186/s12870-024-05114-4 -
Scientific Reports May 2024The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative...
The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4 T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.
Topics: Humans; 3' Untranslated Regions; Lymphocyte Activation; Polyadenylation; CD4-Positive T-Lymphocytes; Gene Expression Regulation; Signal Transduction; RNA-Binding Proteins; CD28 Antigens; T-Lymphocytes
PubMed: 38745101
DOI: 10.1038/s41598-024-61951-1 -
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 -
Nucleus (Austin, Tex.) Dec 2024Paraspeckles are non-membranous subnuclear bodies, formed through the interaction between the architectural long non-coding RNA (lncRNA) nuclear paraspeckle assembly... (Review)
Review
Paraspeckles are non-membranous subnuclear bodies, formed through the interaction between the architectural long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) and specific RNA-binding proteins, including the three (DBHS) family members (PSPC1 (Paraspeckle Component 1), SFPQ (Splicing Factor Proline and Glutamine Rich) and NONO (Non-POU domain-containing octamer-binding protein)). Paraspeckle components were found to impact viral infections through various mechanisms, such as induction of antiviral gene expression, IRES-mediated translation, or viral mRNA polyadenylation. A complex involving NEAT1 RNA and paraspeckle proteins was also found to modulate interferon gene transcription after nuclear DNA sensing, through the activation of the cGAS-STING axis. This review aims to provide an overview on how these elements actively contribute to the dynamics of viral infections.
Topics: Humans; Virus Diseases; Animals; RNA, Long Noncoding; RNA-Binding Proteins
PubMed: 38717150
DOI: 10.1080/19491034.2024.2350178