#m6A modification

[biotech-news-feed]

In the years since the first demonstration that clearance of senescent cells produces rejuvenation in old mice, and the first programs to develop senolytic drugs that can selectively destroy senescent cells, ever more research has been directed into #BioTech #science

[healthcaremarketanalysis]

Exploring RNA Analysis Methods: Techniques for Comprehensive Understanding of RNA

RNA analysis is a cornerstone of molecular biology, enabling researchers to decode the various functions and regulatory mechanisms of RNA in cellular processes. With growing interest in transcriptomics, RNA analysis methods have evolved to offer more precise, high-throughput, and comprehensive insights into gene expression, alternative splicing, RNA modifications, and more. Here, we explore several RNA analysis methods that have become essential tools in biological and medical research.

Download PDF Brochure

1. RNA Sequencing (RNA-Seq)

RNA sequencing is the gold standard for transcriptome analysis. It allows researchers to examine both coding and non-coding RNA with high resolution. RNA-Seq provides quantitative data on gene expression levels, alternative splicing events, and even RNA-editing phenomena. This method has the advantage of being unbiased, offering a comprehensive snapshot of the entire transcriptome.

Steps Involved:

RNA extraction

cDNA synthesis

Sequencing via next-generation sequencing platforms

Data analysis using bioinformatics tools to map reads to reference genomes and quantify expression

2. Quantitative PCR (qPCR)

Quantitative PCR is a highly sensitive method to measure RNA expression levels. It is often used to validate results from RNA-Seq or microarray studies. By amplifying specific RNA sequences and using fluorescent probes, qPCR provides real-time quantification of RNA molecules, offering highly accurate and reproducible data.

Advantages:

High sensitivity

Quantitative results in real time

Often used for validation of gene expression studies

3. Microarrays

Microarray technology allows the simultaneous analysis of thousands of RNA molecules. Although it has been somewhat replaced by RNA-Seq due to the latter’s higher resolution and broader coverage, microarrays remain popular for focused studies on specific genes or pathways. They are relatively inexpensive and easy to use for researchers looking for rapid gene expression profiling.

Key Applications:

Gene expression profiling

Comparative studies across different samples or conditions

Focused analysis of known RNA sequences

4. Northern Blotting

Northern blotting is a classical technique used to detect specific RNA molecules within a mixture of RNA. While it is less commonly used today, northern blotting remains a reliable tool for detecting the presence and size of RNA molecules. This method is particularly useful for validating the results of RNA-Seq or qPCR.

Request Sample Pages

Process Overview:

RNA extraction and electrophoresis

Transfer of RNA onto a membrane

Hybridization with labeled probes specific to the RNA of interest

Detection via autoradiography or chemiluminescence

5. Single-Cell RNA Sequencing (scRNA-Seq)

Single-cell RNA sequencing is a cutting-edge technique that enables researchers to study gene expression at the resolution of individual cells. This method has revolutionized the field of transcriptomics by revealing cellular heterogeneity and identifying rare cell types that might be missed by bulk RNA-Seq.

Advantages:

High resolution for detecting cell-to-cell variability

Crucial for understanding complex tissues and diseases like cancer

Insights into cellular differentiation and development

6. RNA Immunoprecipitation (RIP)

RNA immunoprecipitation is used to study RNA-protein interactions. Researchers use specific antibodies to target RNA-binding proteins, isolating the associated RNA molecules. RIP is particularly valuable in studying RNA modifications, such as methylation, and understanding how RNA-protein complexes influence gene expression.

Applications:

Studying RNA modifications (e.g., m6A methylation)

Understanding the role of RNA-binding proteins in disease

Functional annotation of RNA molecules

7. In Situ Hybridization (ISH)

In situ hybridization is a method used to detect specific RNA sequences in fixed tissue sections or cells. This method provides spatial information about RNA localization within tissues, making it invaluable for developmental biology and cancer research.

Benefits:

Visualization of RNA expression patterns in intact tissues

High spatial resolution

Useful in identifying RNA localization in specific cell types

Conclusion

The diversity of RNA analysis methods allows researchers to study the complex roles of RNA in gene regulation, cellular function, and disease. While RNA-Seq remains the most comprehensive approach, each method offers distinct advantages depending on the research question and experimental needs. By combining these methods, scientists can gain a holistic view of RNA biology, paving the way for advancements in precision medicine and therapeutic development.

Whether it's detecting subtle changes in gene expression or unraveling RNA-protein interactions, these RNA analysis techniques continue to enhance our understanding of the molecular underpinnings of life.

Content Source:

[leedsomics]

Subcellular Level Spatial Transcriptomics with PHOTON

The subcellular localization of RNA is closely linked to its function. Many RNA species are partitioned into organelles and other subcellular compartments for storage, processing, translation, or degradation. Thus, capturing the subcellular spatial distribution of RNA would directly contribute to the understanding of RNA functions and regulation. Here, we present PHOTON (Photoselection of Transcriptome over Nanoscale), a method which combines high resolution imaging with high throughput sequencing to achieve spatial transcriptome profiling at subcellular resolution. We demonstrate PHOTON as a versatile tool to accurately capture the transcriptome of target cell types in situ at the tissue level such as granulosa cells in the ovary, as well as RNA content within subcellular compartments such as the nucleolus and the stress granule. Using PHOTON, we also reveal the functional role of m6A modification on mRNA partitioning into stress granules. These results collectively demonstrate that PHOTON is a flexible and generalizable platform for understanding subcellular molecular dynamics through the transcriptomic lens. http://dlvr.it/TDFjkZ

[rnomics]

Biomedicines, Vol. 12, Pages 2001: Epigenetic Regulation of DNA Methylation and #RNA Interference in Gastric #cancer: A 2024 Update

Gastric #cancer (GC) remains a significant public health concern because of its lethality, underscoring the need for deeper insights into its molecular mechanisms. Recent studies have increasingly highlighted the role of epigenetic modifications as critical players in #cancer progression. Despite their importance, research specifically addressing epigenetic factors in GC is relatively scarce. This paper seeks to bridge that gap by examining recent literature that elucidates the epigenetic landscape associated with GC. The investigation of long noncoding #RNAs (l#ncRNAs) has revealed their substantial involvement in gene dysregulation and epigenetic alterations within GC tumors. Notably, l#ncRNAs such as LINC00853 and LINC01266 have been identified as significant contributors to the epigenetic modulation of gene expression. Furthermore, the overexpression of KAT5 and GPX4 has been shown to mitigate the antiproliferative effects resulting from the depletion of circRHOT1, suggesting a complex interplay between these molecules in GC pathophysiology. Another pivotal aspect of epigenetic regulation in GC involves modifications in N6-methyladenosine (m6A), which play crucial roles in #mRNA maturation processes such as splicing, export, degradation, and translation. m6A modifications are known for their influence on various #cancer-related pathways, thus presenting a potential avenue for targeted interventions. Our findings indicate that the most pronounced instances of epigenetic dysregulation in GC can be traced back to the effects of long l#ncRNAs and alterations in m6A modification patterns. This underscores the urgent need for comprehensive investigations into these epigenetic factors, as a deeper understanding could lead to enhanced diagnostic markers and innovative therapeutic strategies. The integration of genetic and epigenetic considerations is essential for advancing the field of GC research. This synthesis of recent findings concerning epigenetic regulation offers valuable insights that could inform future studies and therapeutic developments. There is a critical need for ongoing research to elucidate the complexities of epigenetic modifications in GC, ultimately improving patient outcomes through tailored interventions. https://www.mdpi.com/2227-9059/12/9/2001?utm_source=dlvr.it&utm_medium=tumblr

[cmlhopeblog]

N6-methyladenosine (m6A) RNA modification’s regulatory role in acute and chronic leukemia

CMLHope.Com http://dlvr.it/TCfcl0

[didanawisgi]

Abstract

The relentless, protracted evolution of the SARS-CoV-2 virus imposes tremendous pressure on herd immunity and demands versatile adaptations by the human host genome to counter transcriptomic and epitranscriptomic alterations associated with a wide range of short- and long-term manifestations during acute infection and post-acute recovery, respectively. To promote viral replication during active infection and viral persistence, the SARS-CoV-2 envelope protein regulates host cell microenvironment including pH and ion concentrations to maintain a high oxidative environment that supports template switching, causing extensive mitochondrial damage and activation of pro-inflammatory cytokine signaling cascades. Oxidative stress and mitochondrial distress induce dynamic changes to both the host and viral RNA m6A methylome, and can trigger the derepression of long interspersed nuclear element 1 (LINE1), resulting in global hypomethylation, epigenetic changes, and genomic instability. The timely application of melatonin during early infection enhances host innate antiviral immune responses by preventing the formation of “viral factories” by nucleocapsid liquid-liquid phase separation that effectively blockades viral genome transcription and packaging, the disassembly of stress granules, and the sequestration of DEAD-box RNA helicases, including DDX3X, vital to immune signaling. Melatonin prevents membrane depolarization and protects cristae morphology to suppress glycolysis via antioxidant-dependent and -independent mechanisms. By restraining the derepression of LINE1 via multifaceted strategies, and maintaining the balance in m6A RNA modifications, melatonin could be the quintessential ancient molecule that significantly influences the outcome of the constant struggle between virus and host to gain transcriptomic and epitranscriptomic dominance over the host genome during acute infection and PASC.

[vehicle-research]

This was an early concept for Viking II. Still quite handsome today.

One of the most difficult problems for limited production car design and a key factor that defines the fundamental shape of a vehicle is the windscreen.

The designer can either borrow an existing production windshield and accept the compromises of that shape and incorporate them into his design, or bite the bullet and design a shape from scratch and then have a custom windshield made, not an inexpensive undertaking.

Cars designed at the VRI went both ways. Viking I used the Toyota E20 (Corolla) windshield that came with the donor car supplied by Toyota. Viking II used a custom shape, the glass being made by a specialist company in Mexico City. The shape was defined by a wire matrix, shipped to Mexico and six months later, a heavily boxed windscreen came back.

The next generation of Vikings would use a single hatch entry system, with the windshield incorporated into the hatch structure. This required a compromise of an aerodynamic shape and low weight (glass is heavy). Vikings IV and V used a cut-down Opel GT windshield, where about 4cm of material was ground off of the lower corners. This allowed a more extreme rake and provided for a more slippery shape, but the yield for the grinding job process was about 3 to 1 (one good windshield achieved out of three grinding attempts). /1

Viking VI used the Opel windscreen, but the cowl shape of the hatch was redesigned to be slightly more upright so the Opel GT windshield could be used without modification. A higher rake is achieved as the lower corners are slightly embedded in the body and then faired in with filler.

Other cars designed around the VRI had similar design choices to make. Bill Brown's B1 and B2 also used an unmodified Opel GT windshield. The Avion uses a second-generation Toyota Celica production windshield.

The next generation of Vikings would move away from the single hatch and so a more aerodynamic, yet economical solution was sought as the cost of custom windshields had risen spectacularly. One day a student at the VRI was thumbing through an old Road and Track and saw an advertisement for a Manta kit car. The Manta was a fairly accurate interpretation of a McLaren M6 GT, but designed to be built on a humble VW beetle or tube frame chassis. Kit prices were kept low, so a Manta windshield was available as a replacement part for a very reasonable price. So Viking VII and VIII used the Manta windscreen.

Time moved on, and the 70's and 80's kit car market slowly declined. After making over 1000 Manta kits, Manta went out of business in 1986 and with it a VRI source of inexpensive aerodynamic windscreens. So going forward, replacement McLaren M6A windscreens had to be sourced at McLaren M6 GT vintage spare prices.

That said, Opel GT windshields are very dear these days, and are available only through enthusiast clubs and cost a pretty penny.

The side windows of Viking cars are made of Abcite coated polycarbonate which can be heat-formed.

You can see the various windscreen shapes in their original applications below:

Opel GT

Manta Montage

McLaren M6 GT

The extreme nature of the Manta windscreen can be appreciated by looking at the Manta's fiberglass molded cockpit frame.

/1 Grinding the corners of a stock Opel GT windshield was about a six-hour process. Grinding the first corner was usually always successful, and now with some process confidence under one's belt, one would throw caution to the wind and a crack would nearly always appear when grinding the second side.

Viking V broke its windshield en route on the SCORE trip. So taken by Viking V, a Detroit auto glass replacement shop's most enthusiastic and youngest employee stayed up all night grinding a new one. He had success on only his second attempt. The nice folks at the shop didn't charge for the first attempt.

[tumimmtxpapers]

Recent Advances in RNA m6A Modification in Solid Tumors and Tumor Immunity

An analogous field to epigenetics is referred to as epitranscriptomics, which focuses on the study of post-transcriptional chemical modifications in RNA. RNA molecules, including mRNA, tRNA, rRNA, and other non-coding RNA molecules, can be edited with numerous modifications. The most prevalent modification in eukaryotic mRNA is N6-methyladenosine (m⁶A), which is a reversible modification found in over 7000 human genes. Recent technological advances have accelerated the characterization of these... http://dlvr.it/T0MYFV

[sisiad]

LncRNA CALML3-AS1 modulated by m6A modification induces BTNL9 methylation to drive non-small-cell lung cancer progression

http://dlvr.it/Sy1Cvl

[ribosome-papers]

Modulation of translational decoding by m6A modification of mRNA

Pubmed: http://dlvr.it/StbJMb

[mindblowingscience]

A human genetic mechanism hijacked by SARS-CoV-2, the coronavirus behind the COVID-19 pandemic, to help it spread also makes it vulnerable to a new class of drug candidates, a new study finds.

Led by researchers at NYU Grossman School of Medicine, a research team showed that coronavirus reproduction in infected human cells requires chemical changes made by the human protein METTL3 to RNA, a key form of genetic material. Additional human proteins involved in the recognition of modified RNA, YTHDF1 and YTHDF3, were also found to be important to the process.

Published online in Genes and Development on June 24, the study showed for the first time that a molecular inhibitor of METTL3, designed by Storm Therapeutics Ltd and called STM2457, dramatically reduced in cell cultures the replication of both pandemic SARS-CoV-2 and, a less severe, seasonal coronavirus, HCoV-OC43, one cause of the common cold.

Continue Reading.

[leedsomics]

Integrative analysis of multi-omics and machine learning highlighted an m6A-related mRNA signature as a robust AAA progression predictor

Objective: Abdominal aortic aneurysm (AAA) is a life-threatening disease in vascular surgery with significant morbidity and mortality rates upon rupture. Despite surgical interventions, effective targeted drugs for non-surgical candidates are lacking. M6A methylation, a dynamic RNA modification, has been implicated in various diseases, but its role in AAA remains poorly understood. In this study, we aimed to explore the participation of M6A in the progression of AAA progression through multi-omics and machine learning. Approach and Results: We conducted methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) to profile the m6A methylome in AAA tissues, identifying differentially methylated genes (DMGs). Integrating multi-omics data from RNA-sequencing (RNA-seq) in GEO databases, we developed a machine learning-based AAA m6A-related mRNA signature (AMRMS) to predict AAA dilation risk. The AMRMS demonstrated robust predictive performance in distinguishing AAA patients with large AAA and small AAA. Notably, the AMRMS highlighted FKBP11 as a key gene with a significant impact on the predicted model. Subsequent single-cell RNA sequencing (ScRNA-seq) revealed the pivotal role of FKBP11-positive plasma cells in AAA progression. Conclusion: Our study provides novel insights into the regulatory role of m6A modification in AAA pathogenesis, and further develop a promising AMRMS for risk evaluation in AAA patients. Furthermore, the identification of FKBP11 positive plasma cells as significant contributors to AAA progression opens new avenues for targeted therapeutic interventions. http://dlvr.it/SwpTgL

[rnomics]

Single-cell discovery of m6A #RNA modifications in the hippocampus [RESEARCH]

N6-Methyladenosine (m6A) is a prevalent and highly regulated #RNA modification essential for #RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m6A is implicated in neurogenesis and learning. Although extensively studied, its presence in specific cell types remains poorly understood. We investigated m6A in the hippocampus at a single-cell resolution, revealing a comprehensive landscape of m6A modifications within individual cells. Through our analysis, we uncovered transcripts exhibiting a dense m6A profile, notably linked to neurological disorders such as Alzheimer's disease. Our findings suggest a pivotal role of m6A-containing transcripts, particularly in the context of CAMK2A neurons. Overall, this work provides new insights into the molecular mechanisms underlying hippocampal physiology and lays the foundation for future studies investigating the dynamic nature of m6A #RNA methylation in the healthy and diseased brain. http://genome.cshlp.org/cgi/content/short/34/6/822?rss=1&utm_source=dlvr.it&utm_medium=tumblr

[cmlhopeblog]

N6-methyladenosine (m6A) RNA modification’s regulatory role in acute and chronic leukemia

CMLHope.Com http://dlvr.it/TCfD4Q