Generating snoRNA-guided Programmable 2'- -methylation.

Small nucleolar RNAs (snoRNAs) are critical in guiding post-transcriptional modifications like 2'- -methylation (Nm), which play crucial roles in downstream processes such as splicing and translation. This study tests a novel method for Nm validation, addressing a significant gap in modern Nm research, and offers insight into the intricacies of snoRNA-guided Nm. While mapping of Nm modifications has seen significant improvement within the past decade, no major techniques have been able to validate these potential sites.

Single-cell mA profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation.

N-methyladenosine (mA) is an abundant mRNA modification in the brain that has important roles in neurodevelopment and brain function. However, because of technical limitations, global profiling of mA sites within the individual cell types that make up the brain has not been possible. Here, we develop a mouse model that enables transcriptome-wide mA detection in any tissue of interest at single-cell resolution.

Early Outcomes in Patients With LVAD Undergoing Heart Transplant via Use of the SherpaPak Cardiac Transport System.

Background: Heart transplant (HT) in recipients with left ventricular assist devices (LVADs) is associated with poor early post-HT outcomes, including primary graft dysfunction (PGD). As complicated heart explants in recipients with LVADs may produce longer ischemic times, innovations in donor heart preservation may yield improved post-HT outcomes. The SherpaPak Cardiac Transport System is an organ preservation technology that maintains donor heart temperatures between 4 °C and 8 °C, which may minimize ischemic and cold-induced graft injuries.

2'--methylation (Nm) in RNA: progress, challenges, and future directions.

RNA 2'--methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm.

Programmable protein expression using a genetically encoded mA sensor.

The N-methyladenosine (mA) modification is found in thousands of cellular mRNAs and is a critical regulator of gene expression and cellular physiology. mA dysregulation contributes to several human diseases, and the mA methyltransferase machinery has emerged as a promising therapeutic target. However, current methods for studying mA require RNA isolation and do not provide a real-time readout of mRNA methylation in living cells.

Metabolomic profiling during ex situ normothermic perfusion before heart transplantation defines patterns of substrate utilization and correlates with markers of allograft injury.

Background: Cardiac metabolism is altered in heart failure and ischemia-reperfusion injury states. We hypothesized that metabolomic profiling during ex situ normothermic perfusion before heart transplantation (HT) would lend insight into myocardial substrate utilization and report on subclinical and clinical allograft dysfunction risk.

Methods: Metabolomic profiling was performed on serial samples of ex situ normothermic perfusate assaying biomarkers of myocardial injury in lactate and cardiac troponin I (TnI) as well as metabolites (66 acylcarnitines, 15 amino acids, nonesterified fatty acids [NEFA], ketones, and 3-hydroxybutyrate).

m6A regulates breast cancer proliferation and migration through stage-dependent changes in Epithelial to Mesenchymal Transition gene expression.

While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer.

One Small Step for a Patient, One Giant Leap for Orthostatic Hypotension.

A 52-year-old man with ischemic cardiomyopathy presented with progressive, severe orthostatic hypotension refractory to medical therapy. Standard abdominal and leg compression devices were used without success. A novel, inflatable abdominal compression device was created that alleviated the patient's symptoms and maintained his blood pressure.

RBM45 is an mA-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

N-methyladenosine (mA) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. mA is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which mA contributes to brain development are incompletely understood.

Improved Methods for Deamination-Based mA Detection.

-methyladenosine (mA) is a critical regulator of gene expression and cellular function. Much of our knowledge of mA has been enabled by the identification of mA sites transcriptome-wide. However, global mA profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making mA profiling from rare cell types or scarce tissue samples infeasible.

Donation After Circulatory Death in Heart Transplantation: History, Outcomes, Clinical Challenges, and Opportunities to Expand the Donor Pool.

Heart transplantation remains the gold-standard therapy for end-stage heart failure; the expected median survival range is 12-13 years. More than 30,000 heart transplants have been performed globally in the past decade alone. With advances in medical and surgical therapies for heart failure, including durable left ventricular assist devices, an increasing number of patients are living with end-stage disease.

Transcriptome-Wide Identification of 2'-O-Methylation Sites with RibOxi-Seq.

The ability to detect 2'-O-methylation sites (Nm) in high-throughput fashion is important, as increasing evidence points to a more diverse landscape for this RNA modification as well as the possibility of yet unidentified functions. Here we describe an optimized version of RibOxi-seq, which is built upon the original published method, that not only accurately profiles ribosomal RNA (rRNA) Nm sites with minimal RNA input but is also robust enough to identify mRNA intronic and exonic sites.

Proteomic profiling identifies CLEC4C expression as a novel biomarker of primary graft dysfunction after heart transplantation.

Purpose: Clinical models to identify patients at high risk of primary graft dysfunction (PGD) after heart transplantation (HT) are limited, and the underlying pathophysiology of this common post-transplant complication remains poorly understood. We sought to identify whether pre-transplant levels of circulating proteins reporting on immune activation and inflammation are associated with incident PGD.

Methods: The study population consisted of 219 adult heart transplant recipients identified between 2016 and 2020 at Duke University Medical Center, randomly divided into derivation (n = 131) and validation (n = 88) sets.

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.

Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear.

Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.

2'-O-methylation (Nm) is an RNA modification commonly found on rRNA and snRNA, and at the mRNA 5'-cap, but has more recently been found internally on mRNA. The study of internal Nm modifications on mRNA is in the early stages, but we have reported that this sort of Nm modification can regulate mRNA abundance and translation. Although there are many methods to determine the presence of Nm on rRNA, detecting Nm on specific mRNA transcripts is technically difficult because they are much less abundant than rRNA.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops.

Noncoding RNAs in Cardiovascular Disease: Current Knowledge, Tools and Technologies for Investigation, and Future Directions: A Scientific Statement From the American Heart Association.

Background: The discovery that much of the non-protein-coding genome is transcribed and plays a diverse functional role in fundamental cellular processes has led to an explosion in the development of tools and technologies to investigate the role of these noncoding RNAs in cardiovascular health. Furthermore, identifying noncoding RNAs for targeted therapeutics to treat cardiovascular disease is an emerging area of research. The purpose of this statement is to review existing literature, offer guidance on tools and technologies currently available to study noncoding RNAs, and identify areas of unmet need.

Altered mA Modification of Specific Cellular Transcripts Affects Flaviviridae Infection.

The RNA modification N-methyladenosine (mA) modulates mRNA fate and thus affects many biological processes. We analyzed mA across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters mA modification of specific cellular transcripts, including RIOK3 and CIRBP.

Epitranscriptomic Addition of mC to HIV-1 Transcripts Regulates Viral Gene Expression.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (mC) and 2'O-methyl modifications being particularly prevalent.

Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo.

Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2'-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression.

Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of -methyladenosine (mA), 5-methylcytosine (mC), and 2'O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of mA and mC residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites.

N-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression.

The mRNA modification N-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor.

Long-range function of secreted small nucleolar RNAs that direct 2'--methylation.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'--methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs.