Integrative MeRIP-Seq and RNA-Seq Analyses Reveal Innate Immune and Infection-Related Transcriptomic Changes upon METTL3 Knockout

Quantitative single-base m6A profiling reveals dynamic reprogramming, evolutionary conservation and transcriptional regulation in bacteria.

N6-methyladenosine (m6A) is a widespread RNA modification that regulates RNA metabolism in eukaryotes, but its distribution and function in bacteria remain poorly defined. Here, we apply GLORI sequencing to generate single-base resolution transcriptome-wide m6A maps in seven bacterial species. We identify 2,845 m6A sites during exponential growth and find extensive condition-dependent methylation dynamics in three strains. In Pseudomonas syringae, m6A remodeling is associated with virulence-related pathways. Comparative analyses reveal 455 conserved m6A site pairs enriched in genes required for growth, energy metabolism, and transmembrane transport. Integrating methylation, transcript abundance, and RNA stability analyses shows that m6A is associated with reduced mRNA abundance and increased RNA stability. We further identify the rRNA methyltransferases RlmF and RlmJ as bacterial mRNA m6A writers. Together, these findings provide a quantitative atlas of bacterial m6A and establish a foundation for understanding its regulatory and evolutionary roles.

Integration of ATAC-seq and RNA-seq analysis identifies host genes related with PRRSV infection

The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious pathogen. Viral infections often enhance their replication by modulating the structure and expression of host genes. However, it remains unclear whether PRRSV employs a similar mechanism to achieve self-replication. To address this question, the current study combined assay for transposase accessible chromatin sequencing (ATAC-seq) and ribonucleic acid (RNA) sequencing (RNA-seq) to identify accessible chromatin regions and key host genes associated with PRRSV infection. By comparing the PRRSV-infected group with the control group, we initially detected 8664 differentially accessible chromatin regions and 4037 differentially expressed genes. Motif analysis of these differential chromatin regions revealed several potential cis-regulatory elements containing binding sites for transcription factors. Further integration of ATAC-seq and RNA-seq results identified 1352 overlapping genes between the PRRSV-infected and control groups. A significant positive correlation between differential gene expression and chromatin accessibility signals suggests that chromatin remodeling may drive transcriptional changes during infection. Protein–protein interaction (PPI) network analysis highlighted candidate genes potentially associated with PRRSV infection in hosts, such as IL1B, CCL20, CXCL10, CSF3, etc. Given their potential association with the infection mechanism, these genes could serve as candidate targets for the future development of prophylactic vaccines and therapeutic strategies. Additionally, several signaling pathways that may regulate immune and inflammatory responses were significantly enriched in our ATAC-seq and RNA-seq analyses. These findings provide valuable insights into the molecular mechanisms underlying PRRSV infection and pave the way for developing more effective preventive and treatment measures.

Unraveling genomic regulatory complexity by adding chromatin accessibility to the long-read sequencing toolkit 2247245

Chromatin structure regulates gene expression, shaping immune cell responses and differentiation. Profiling accessibility, transcription factor binding, and DNA methylation is key to understanding immune regulation. Short-read sequencing (SRS) methods like ATAC-seq and ChIP-seq provide valuable insights but lack resolution and cannot capture multiple chromatin features together, limiting our ability to define their coordinated roles in immune function. We optimized Fiber-seq, a multiomic long-read sequencing (LRS) method to simultaneously profile chromatin accessibility and endogenous methylation using LRS. Fiber-seq uses a non-specific DNA methyltransferase to label accessible DNA by creating N6-methyladenosine, a mark rarely found in eukaryotic genomes. This rapid enzymatic incubation also preserves endogenous DNA methylation so that both methylation marks can be detected by direct LRS. We validated this new method using human lymphoblast cells (K562). Aggregate Fiber-seq accessibility profiles were highly concordant with published ATAC-seq datasets. We confirmed that 6mA-labeling preserved sequencing accuracy and detection of CpG methylation. We also found nucleosome and protein footprints could be inferred from Fiber-seq data. We have validated multiple transcription factor inferred footprints are highly concordant with published CUT&RUN or ChIP-seq datasets. Further, these footprints are captured at a per-molecule and near-base pair resolution, uncovering multiple binding events in close proximity, which is usually obscured by lower resolution SRS-based methods. Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variation on single molecules at near—base-pair resolution. This integrated view reveals how genetic and epigenetic features interact to regulate gene expression and provides a powerful new framework for dissecting immune cell function and disease mechanisms. NIH R44 GM148145 Technological Innovations in Immunology (TECH)

Epitranscriptomic Regulation of mRNA Stability in Plants: Roles of m6A, m5C, and Pseudouridine

m5C Methylation of mRNA: Still More Questions Than Answers

mRNA plays a pivotal role in cellular processes of genetic information transfer, and post-transcriptional modifications of its nucleotides enable regulation of these processes with each particular mRNA. m5C methylation is a specific RNA modification that is rather common in tRNA, rRNA, lncRNA and other types of non-coding RNAs, whereas in mRNA it is found not very often. However, the functioning of m5C-methylated mRNAs differs from the non-methylated ones quite dramatically. Of the eight m5C RNA methyltransferases in humans, only two, NSUN2 and NSUN6, were found to be capable of modifying the main portion of cellular mRNAs. A deficiency of NSUN2 and NSUN6 causes global changes in the transcriptome and translatome, and a corruption of the NSUN2 gene in humans is associated with neurodegenerative diseases and intellectual disability. Despite intensive research of m5C mRNA methylation in recent years, many aspects of this phenomenon and its significance remain problematic and far from understood. In this review, we discuss the available information on mRNA methylome biogenesis, methods for its study and analysis, effects of m5C modification on mRNA life, and place it in the general context of cell biology, attempting to draw the biological relevance of m5C mRNA methylation. We highlight the main inconsistencies and difficulties that arise, analyze their possible causes, and propose potential directions for further research that could clarify the controversial issues and provide a link between the NSUN2 deficiency and neurodegenerative diseases.

Mitochondrial RNA modifications in gene expression and cancer biology.

RNA modifications are essential in regulating gene expression at the post-transcriptional level. Recent studies, including our own, have highlighted that RNA modifications, such as N6-methyladenosine (m6A) and methyl-5-cytosine (m5C), play a crucial role in tumorigenesis, metabolism, and anti-tumor immunity. Targeting RNA modification machinery may represent a promising therapeutic strategy in cancer. Intriguingly, emerging evidence reveals numerous modifications in mitochondrial RNA (mt-RNA), expanding the concept of epitranscriptomics to mitochondria. The mammalian mitochondrion possesses its own genome, which encodes 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and 13 proteins necessary for energy production via oxidative phosphorylation (OxPhos). The mitochondrial transcriptome is produced from large polycistronic transcripts, implying that mitochondrial gene expression is predominantly regulated post-transcriptionally. In this review, we summarize all currently known mt-RNA modifications, their potential regulatory machinery, as well as their biological functions in tumorigenesis and metabolism. Additionally, given that this field is still in its infancy, we discuss several critical knowledge gaps and propose future research directions to clarify the mechanistic and clinical significance in the study of mt-RNA modifications.