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1.
Nat Commun ; 12(1): 5981, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645812

RESUMO

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.

2.
Methods Enzymol ; 658: 25-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517949

RESUMO

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven ß-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of m7G, m3C, D, and ho5C in variety of cellular RNAs, including in particular rRNAs and tRNAs.


Assuntos
Citidina , Guanosina , Citidina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA de Transferência/genética , Análise de Sequência de RNA
3.
Hum Genet ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545459

RESUMO

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

4.
Adv Biol (Weinh) ; 5(10): e2100866, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34535986

RESUMO

Epitranscriptomics heavily rely on chemical reagents for the detection, quantification, and localization of modified nucleotides in transcriptomes. Recent years have seen a surge in mapping methods that use innovative and rediscovered organic chemistry in high throughput approaches. While this has brought about a leap of progress in this young field, it has also become clear that the different chemistries feature variegated specificity and selectivity. The associated error rates, e.g., in terms of false positives and false negatives, are in large part inherent to the chemistry employed. This means that even assuming technically perfect execution, the interpretation of mapping results issuing from the application of such chemistries are limited by intrinsic features of chemical reactivity. An important but often ignored fact is that the huge stochiometric excess of unmodified over-modified nucleotides is not inert to any of the reagents employed. Consequently, any reaction aimed at chemical discrimination of modified versus unmodified nucleotides has optimal conditions for selectivity that are ultimately anchored in relative reaction rates, whose ratio imposes intrinsic limits to selectivity. Here chemical reactivities of canonical and modified ribonucleosides are revisited as a basis for an understanding of the limits of selectivity achievable with chemical methods.

5.
Methods ; 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34481083

RESUMO

Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple "mock-treated" controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific reagent, already extensively used in the past for RNA sequencing and structural probing. Hydrazine is highly reactive to U and shows low reaction rates with ψ residues, allowing their distinction by deep sequencing-based protocols. However, other modified RNA residues also show particular behavior upon hydrazine treatment. Here we present methodological developments allowing to use HydraPsiSeq for precise quantification of RNA pseudouridylation and also detection and quantification of some other RNA modifications, in addition to ψ residues.

6.
Prog Neurobiol ; 205: 102118, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34245849

RESUMO

tRNA-derived small RNAs (tsRNA) are a recently identified family of non-coding RNA that have been associated with a variety of cellular functions including the regulation of protein translation and gene expression. Recent sequencing and bioinformatic studies have identified the broad spectrum of tsRNA in the nervous system and demonstrated that this new class of non-coding RNA is produced from tRNA by specific cleavage events catalysed by ribonucleases such as angiogenin and dicer. Evidence is also accumulating that production of tsRNA is increased during disease processes where they regulate stress responses, proteostasis, and neuronal survival. Mutations to tRNA cleaving and modifying enzymes have been implicated in several neurodegenerative disorders, and tsRNA levels in the blood are advancing as biomarkers for neurological disease. In this review we summarize the physiological importance of tsRNA in the central nervous system and their relevance to neurological disease.

7.
Methods Mol Biol ; 2298: 77-95, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085239

RESUMO

Epitranscriptomics is an emerging field where the development of high-throughput analytical technologies is essential to profile the dynamics of RNA modifications under different conditions. Despite important advances during the last 10 years, the number of RNA modifications detectable by next-generation sequencing is restricted to a very limited subset. Here, we describe a highly efficient and fast method called AlkAniline-Seq to map simultaneously two different RNA modifications: 7-methyl-guanosine (m7G) and 3-methyl-cytosine (m3C) in RNA. Our protocol is based on three subsequent chemical/enzymatic steps allowing the enrichment of RNA fragments ending at position n + 1 to the modified nucleotide, without any prior RNA selection. Therefore, AlkAniline-Seq demonstrates an outstanding sensitivity and specificity for these two RNA modifications. We have validated AlkAniline-Seq using bacterial, yeast, and human total RNA, and here we present, as an example, a synthetic view of the complete profiling of these RNA modifications in S. cerevisiae tRNAs.


Assuntos
Citosina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Linhagem Celular , Citosina/metabolismo , Guanosina/genética , Células HEK293 , Humanos , Metilação , Sensibilidade e Especificidade , Análise de Sequência de RNA/métodos
8.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145251

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética
9.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
10.
Nucleic Acids Res ; 49(10): 5568-5587, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33999208

RESUMO

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin.


Assuntos
DNA/metabolismo , Heterocromatina , RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Metilação , Camundongos , Células-Tronco Embrionárias Murinas , Sequências de Repetição em Tandem
11.
Cancers (Basel) ; 13(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919717

RESUMO

The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide.

12.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498319

RESUMO

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells-especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell-cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1-2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHCP using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.


Assuntos
Melanoma/imunologia , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Citometria de Fluxo/métodos , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Imuno-Histoquímica/métodos , Imunofenotipagem/métodos , Melanoma/patologia , Camundongos , Pessoa de Meia-Idade , Transgenes
13.
Genes (Basel) ; 12(1)2021 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-33435213

RESUMO

Analysis of RNA by deep-sequencing approaches has found widespread application in modern biology. In addition to measurements of RNA abundance under various physiological conditions, such techniques are now widely used for mapping and quantification of RNA modifications. Transfer RNA (tRNA) molecules are among the frequent targets of such investigation, since they contain multiple modified residues. However, the major challenge in tRNA examination is related to a large number of duplicated and point-mutated genes encoding those RNA molecules. Moreover, the existence of multiple isoacceptors/isodecoders complicates both the analysis and read mapping. Existing databases for tRNA sequencing provide near exhaustive listings of tRNA genes, but the use of such highly redundant reference sequences in RNA-seq analyses leads to a large number of ambiguously mapped sequencing reads. Here we describe a relatively simple computational strategy for semi-automatic collapsing of highly redundant tRNA datasets into a non-redundant collection of reference tRNA sequences. The relevance of the approach was validated by analysis of experimentally obtained tRNA-sequencing datasets for different prokaryotic and eukaryotic model organisms. The data demonstrate that non-redundant tRNA reference sequences allow improving unambiguous mapping of deep sequencing data.


Assuntos
Bacillus subtilis/genética , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , RNA Bacteriano/genética , RNA de Transferência/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA
14.
EMBO J ; 40(6): e105496, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33283887

RESUMO

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.

15.
Nucleic Acids Res ; 49(4): e23, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33313868

RESUMO

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.


Assuntos
Adenosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/química , Análise de Sequência de RNA/métodos , Adenosina/análise , Algoritmos , Animais , Cromatografia Líquida , Desaminação , Drosophila melanogaster/genética , Células HEK293 , Células HeLa , Humanos , RNA Longo não Codificante/química , RNA Mensageiro/química , RNA Ribossômico 18S/química , Alinhamento de Sequência , Espectrometria de Massas em Tandem
16.
RNA Biol ; 18(5): 696-708, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33356825

RESUMO

Beyond their high clinical relevance worldwide, flaviviruses (comprising dengue and Zika viruses) are of particular interest to understand the spatiotemporal control of RNA metabolism. Indeed, their positive single-stranded viral RNA genome (vRNA) undergoes in the cytoplasm replication, translation and encapsidation, three steps of the flavivirus life cycle that are coordinated through a fine-tuned equilibrium. Over the last years, RNA methylation has emerged as a powerful mechanism to regulate messenger RNA metabolism at the posttranscriptional level. Not surprisingly, flaviviruses exploit RNA epigenetic strategies to control crucial steps of their replication cycle as well as to evade sensing by the innate immune system. This review summarizes the current knowledge about vRNA methylation events and their impacts on flavivirus replication and pathogenesis. We also address the important challenges that the field of epitranscriptomics faces in reliably and accurately identifying RNA methylation sites, which should be considered in future studies on viral RNA modifications.

17.
Nucleic Acids Res ; 48(22): 12833-12844, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33275131

RESUMO

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2'-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.


Assuntos
Imunidade Inata/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Receptor 7 Toll-Like/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Guanosina/genética , Guanosina/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Metilação , Processamento Pós-Transcricional do RNA/imunologia , RNA de Transferência/imunologia , Receptor 7 Toll-Like/imunologia
18.
Cells ; 9(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003620

RESUMO

Lipid exchange among biological membranes, lipoprotein particles, micelles, and liposomes is an important yet underrated phenomenon with repercussions throughout the life sciences. The premature loss of lipid molecules from liposomal formulations severely impacts therapeutic applications of the latter and thus limits the type of lipids and lipid conjugates available for fine-tuning liposomal properties. While cholesterol derivatives, with their irregular lipophilic surface shape, are known to readily undergo lipid exchange and interconvert, e.g., with serum, the situation is unclear for lipids with regular, linear-shaped alkyl chains. This study compares the propensity of fluorescence-labeled lipid conjugates of systematically varied lengths to migrate from liposomal particles consisting mainly of egg phosphatidyl choline 3 (EPC3) and cholesterol into biomembranes. We show that dialkyl glyceryl lipids with chains of 18-20 methylene units are inherently stable in liposomal membranes. In contrast, C16 lipids show some lipid exchange, albeit significantly less than comparable cholesterol conjugates. Remarkably, the C18 chain length, which confers noticeable anchor stability, corresponds to the typical chain length in biological membranes.


Assuntos
Química Click/métodos , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Lipossomos/química , Linhagem Celular Tumoral , Difusão Dinâmica da Luz , Citometria de Fluxo , Glicerol/química , Humanos , Lipídeos/análise , Lipossomos/síntese química , Membranas Artificiais , Microscopia de Fluorescência , Polímeros/química
19.
Nucleic Acids Res ; 48(19): e110, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32976574

RESUMO

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.


Assuntos
Pseudouridina/isolamento & purificação , RNA Mensageiro , RNA Ribossômico , RNA de Transferência , Análise de Sequência de RNA/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais , Saccharomyces cerevisiae/genética
20.
Genes (Basel) ; 11(8)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824672

RESUMO

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn2+ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m1A, m22G, m1G and m3C showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn2+ on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis.


Assuntos
Íons/metabolismo , Manganês/metabolismo , Transcrição Reversa , Pareamento de Bases , Íons/química , Manganês/química , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleosídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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