Interconnections between m6A RNA modification, RNA structure, and protein-RNA complex assembly.

Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein-RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N6-methyladenosine (m6A) is the most common base modification on mRNAs and regulatory noncoding RNAs and has been shown to impact RNA structure and directly modulate protein-RNA interactions. In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 m6A writer complex is specifically targeted to RNA assisted by protein-RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the m6A modification affects RNA folding and protein-RNA interactions.

Quantification of Poly(A) Tail Length and Terminal Modifications Using Direct RNA Sequencing.

Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts' poly(A) tails and detect the presence of terminal guanylation and uridylation.

Measuring Transcription Dynamics of Individual Genes Inside Living Cells.

Transcription is a highly dynamic process, which, for many genes, occurs in stochastic bursts. Studying what regulates these stochastic bursts requires visualization and quantification of transcription dynamics in single living cells. Such measurements of bursting can be accomplished by labeling nascent transcripts of single genes fluorescently with the MS2 and PP7 RNA labeling techniques. Live-cell single-molecule microscopy of transcription in real time allows for the extraction of transcriptional bursting kinetics inside single cells. This chapter describes how to set up the MS2 or PP7 RNA labeling system of endogenous genes in both budding yeast (Saccharomyces cerevisiae) and mammalian cells (mouse embryonic stem cells). We include how to genetically engineer the cells with the MS2 and PP7 system, describe how to perform the live-microscopy experiments and discuss how to extract transcriptional bursting parameters of the genes of interest.

A FRET-Based Assay and Computational Tools to Quantify Enzymatic Rates and Explore the Mechanisms of RNA Deadenylases in Heterogeneous Environments.

We developed a medium-throughput assay that can measure the time-dependent distribution of RNA products generated as a deadenylase degrades a polyadenosine (poly(A)) RNA tract, thereby providing insight into the mechanism of deadenylation. Importantly, this assay can be performed in both homogeneous and heterogeneous environments without relying on gel electrophoresis of RNA products or coupled enzymatic reactions that indirectly report on the RNA distribution through the detection of freed adenosine monophosphate. In parallel, we have established an open-source, Python-based command-line software package, deadenylationkinetics, that can be used to numerically simulate and/or fit the datasets afforded by our assay with different deadenylation mechanisms to determine the most likely case and estimate the associated rate constants. In this chapter, we detail the implementation of our method and the quantification of poly(A) RNA binding and degradation kinetics in application to a truncated version of CNOT7 from the CCR4-NOT deadenylation complex, which serves as a model deadenylase with enhanced activity.

Transcriptome-Wide Analysis of mRNA Adenylation Status in Yeast Using Nanopore Sequencing.

There are multiple methods for studying deadenylation, either in vitro or in vivo, which allow for observation of mRNA abundance or poly(A) tail dynamics. However, direct RNA sequencing using the Oxford Nanopore Technologies (ONT) platform makes it possible to conduct transcriptome-wide analyses at the single-molecule level without the PCR bias introduced by other methods. In this chapter, we provide a protocol to measure both RNA levels and poly(A)-tail lengths in the yeast Saccharomyces cerevisiae using ONT.

Measuring Poly-Adenosine Tail Length of RNAs by High-Resolution Northern Blotting Coupled with RNase H Cleavage.

The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points.

An RNA-Ligation-Based RACE-PAT Assay to Monitor Poly(A) Tail Length of mRNAs of Interest.

In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End-Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3' ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5' extension. The choice of the oligonucleotide 3' end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5' extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost.

RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.

Roles for Bile Acid Signaling and Nonsense-Mediated Ribonucleic Acid Decay in Small Bowel Resection-Associated Liver Injury.

INTRODUCTION: Massive intestinal loss resulting in short bowel syndrome has been linked to intestinal failure associated liver disease. Efforts to elucidate the driving force behind the observed hepatic injury have identified inflammatory mediators, alterations in the microbiome, extent of structural and functional intestinal adaptation, and toxic shifts in the bile acid pool. In the present study, we posit that ileocecal resection interrupts the delivery of these hepatotoxic substances to the liver by physically disrupting the enterohepatic circulation, thereby shielding the liver from exposure to the aforementioned noxious stimuli. METHODS: Mice underwent sham, 50% proximal, or 50% distal small bowel resection (SBR), with or without tauroursodeoxycolic acid supplementation. Enterohepatic signaling and nonsense-mediated ribonucleic acid (RNA) decay were evaluated and correlated with hepatic injury. RESULTS: When compared to 50% proximal SBR, mice that underwent ileocecal resection exhibited reduced hepatic oxidative stress and exhibited a more physiological bile acid profile with increased de novo bile acid synthesis, enhanced colonic bile acid signaling, and reduced hepatic proliferation. Distal intestinal resection promoted an adaptive response including via the nonsense-mediated RNA decay pathway to satisfactorily process injurious messenger RNA and successfully maintain homeostasis. By contrast, this adaptive response was not observed in the proximal SBR group and hepatic injury persisted. CONCLUSIONS: In summary, interruption of enterohepatic circulation via ileocecal resection abrogates the liver's exposure to toxic and inflammatory mediators while promoting physiological adaptations in bile acid metabolism and maintaining existing homeostatic pathways.

K-Homology Type Splicing Regulatory Protein: Mechanism of Action in Cancer and Immune Disorders.

K homology-type splicing regulatory protein (KSRP) is emerging as a key player in cancer biology, and immunology. As a single-strand nucleic acid binding protein it functions in both transcriptional and post-transcriptional regulation, while facilitating multiple stages of RNA metabolism to affect proliferation and control cell fate. However, it must interact with other proteins to determine the fate of its bound substrate. Here we provide an minireview of this important regulatory protein and describe its complex subcellular functions to affect RNA metabolism, stability, miRNA biogenesis and maturation, stress granule function, metastasis, and inflammatory processes.

METTL3 and METTL14 regulate IL-6 expression via RNA m6A modification of zinc transporter SLC39A9 and DNA methylation of IL-6 in periodontal ligament cells.

The inflammatory response is a key process in periodontitis. The N6-methyladenosine (m6A) modification has been proven to be involved in various physiological and pathological processes. This study aims to investigate the role and downstream mechanism of N6-adenosine-enzyme subunits methyltransferase (METTL) 3 and 14 in the inflammatory response of periodontal ligament cells (PDLCs). The total m6A content and the expression of METTL3 and METTL14 were upregulated in lipopolysaccharide (LPS)-stimulated PDLCs. Knockdown of METTL3 or METTL14 suppressed the LPS-induced interleukin (IL)-6 expression, as shown by quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Mechanistically, conjoint analysis of m6A sequencing of METTL3-knockdown and METTL14-knockdown PDLCs revealed that the expression of solute carrier family 39 member 9 (SLC39A9) was mediated in a m6A-dependent manner. The suppression of LPS-induced IL-6 by METTL3 or METTL14 knockdown was partially counteracted by SLC39A9 knockdown, which induced downregulation of intracellular zinc via immunofluorescence staining. Amplicon bisulfite sequencing (AmpBS) demonstrated that METTL3/14 knockdown increased the methylation at one position of the IL-6 promoter, while SLC39A9 knockdown decreased it, which was basically consistent with the intracellular zinc concentration and negatively associated with IL-6 expression. Moreover, METTL3 or METTL14 knockdown attenuated the LPS-induced phosphorylation of p38 and JNK mitogen-activated protein kinase (MAPK), which was partially counteracted by SLC39A9 knockdown. These results revealed the "LPS-METTL3/14-SLC39A9-zinc-IL-6" axis and involvement of p38 and JNK MAPK signaling pathway in the inflammatory responses of PDLCs.

Nucleolus imaging based on naphthalimide derivatives.

Nucleolus was an important cellular organelle. The abnormal morphology and number of the nucleolus have been considered as diagnostic biomarkers for some human diseases. However, the imaging agent based on nucleolus was limited. In this manuscript, a series of nucleolar fluorescent probes based on naphthalimide derivatives (NI-1 âˆ¼ NI-5) had been designed and synthesized. NI-1 âˆ¼ NI-5 could penetrate cell membranes and nuclear membranes, achieve clear nucleolar staining in living cells. These results suggested that the presence of amino groups on the side chains of naphthalimide backbone could enhance the targeting to the cell nucleolus. In addition, the molecular docking results showed that NI-1 âˆ¼ NI-5 formed hydrogen bonds and hydrophobic interactions with RNA, and exhibited enhanced fluorescence upon binding with RNA. These results will provide favorable support for the diagnosis and treatment of nucleolus-related diseases in the future.

Simultaneous extraction and detection of DNA and RNA from viruses, prokaryotes, and eukaryotes in wastewater using a modified COPMAN.

Wastewater surveillance can offer a comprehensive grasp of infectious disease prevalence and human health because wastewater contains various human-derived microbial pathogens, including viruses, bacteria, and fungi. However, methods capable of simultaneous detection of multiple groups of targets in the automated systems and large-scale surveillance are still under development. Here, we demonstrated the modification, involving the addition of bead-beating, to the existing COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater) enabled enhanced detection of various microorganisms, including SARS-CoV-2. The modified method, termed bead-beating COPMAN (BB-COPMAN), was evaluated through spike-and-recovery experiments and comparative analysis against three previously reported methods for simultaneous DNA/RNA detection. Our study targeted a range of microorganisms, including enveloped and non-enveloped RNA viruses (SARS-CoV-2, PMMoV), a DNA virus (crAssphage), archaea, gram-negative and gram-positive bacteria (E. coli, Lachnospiraceae), antibiotic resistance gene (ampC), and fungi (Candida albicans). The recovery rates of BB-COPMAN for gram-negative and gram-positive bacteria were 17 and 2.1-fold higher, respectively, compared to the method for DNA/RNA detection. Additionally, BB-COPMAN exhibited the highest extraction efficiency among the tested methods, achieving 1.2-5.7 times more DNA and 1.1-69 times more RNA yield on average. BB-COPMAN allowed the detection of SARS-CoV-2 from all nine samples and PMMoV at concentrations 39-97 times higher than other methods. Moreover, BB-COPMAN detected larger amounts of DNA for four out of six DNA targets than the previously reported DNA/RNA detection method. These results demonstrated that BB-COPMAN enables enhanced detection of multiple targets in a single flow of nucleic acid extraction, making the method well-suited for automated systems. In conclusion, BB-COPMAN is a promising method in wastewater surveillance for assessing the prevalence of wide range of pathogenic microorganisms.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

Immune checkpoints signature-based risk stratification for prognosis of patients with gastric cancer.

Until now, few researches have comprehensive explored the role of immune checkpoints (ICIs) and tumor microenvironment (TME) in gastric cancer (GC) patients based on the genomic data. RNA-sequence data and clinical information were obtained from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) database, GSE84437 and GSE84433. Univariate Cox analysis identified 60 ICIs with prognostic values, and these genes were then subjected to NMF cluster analysis and the GC samples (n = 804) were classified into two distinct subtypes (Cluster 1: n = 583; Cluster 2: n = 221). The Kaplan-Meier curves for OS analysis indicated that C1 predicted a poorer prognosis. The C2 subtype illustrated a relatively better prognosis and characteristics of "hot tumors," including high immune score, overexpression of immune checkpoint molecules, and enriched tumor-infiltrated immune cells, indicating that the NMF clustering in GC was robust and stable. Regarding the patient's heterogeneity, an ICI-score was constructed to quantify the ICI patterns in individual patients. Moreover, the study found that the low ICI-score group contained mostly MSI-low events, and the high ICI-score group contained predominantly MSI-high events. In addition, the ICI-score groups had good responsiveness to CTLA4 and PD-1 based on The Cancer Immunome Atlas (TCIA) database. Our research firstly constructed ICIs signature, as well as identified some hub genes in GC patients.

The implications of physiological biomolecular condensates in amyotrophic lateral sclerosis.

In recent years, there has been an emphasis on the role of phase-separated biomolecular condensates, especially stress granules, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). This is largely due to several ALS-associated mutations occurring in genes involved in stress granule assembly and observations that pathological inclusions detected in ALS patient neurons contain stress granule proteins, including the ALS-linked proteins TDP-43 and FUS. However, protein components of stress granules are also found in numerous other phase-separated biomolecular condensates under physiological conditions which are inadequately discussed in the context of ALS. In this review, we look beyond stress granules and describe the roles of TDP-43 and FUS in physiological condensates occurring in the nucleus and neurites, such as the nucleolus, Cajal bodies, paraspeckles and neuronal RNA transport granules. We also discuss the consequences of ALS-linked mutations in TDP-43 and FUS on their ability to phase separate into these stress-independent biomolecular condensates and perform their respective functions. Importantly, biomolecular condensates sequester multiple overlapping protein and RNA components, and their dysregulation could contribute to the observed pleiotropic effects of both sporadic and familial ALS on RNA metabolism.

Niujiao Dihuang Jiedu decoction promotes SLC7A11 m5C methylation modification against ferroptosis in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) constitutes a prevalent manifestation of liver failure within clinical settings. This condition manifests swiftly and is characterized by an exceedingly elevated fatality rate. OBJECTIVE: While numerous investigations have delved into the role of RNA methylation in ferroptosis, the impact of such methylation on ACLF-associated ferroptosis remains notably underexplored. This study aimed to elucidate the molecular mechanism underlying the efficacy of Niujiao Dihuang Jiedu decoction (NDD) in mitigating ferroptosis in ACLF, with a specific focus on RNA 5-methylcytosine (m5C) methylation. MATERIALS AND METHODS: An ACLF rat model was established alongside an erastin-induced ferroptosis model in LO2 cells. Both in vitro and in vivo experiments were conducted to substantiate NDD's influence on ferroptosis. The modifying influence of methylase NOL1/NOP2/sun domain (NSUN5) upon SLC7A11, a key ferroptosis-associated gene, was probed through dot blot, immunofluorescence co-localization, and RNA binding protein immunoprecipitation (RIP) experiments. RESULTS: Serological and hepatic histopathological findings indicated NDD's discernible therapeutic impact on ACLF. Furthermore, ferroptosis phenotype experiments revealed NDD's proficiency in effectively impeding the occurrence and development of ferroptosis. Dot blot assays demonstrated a reduction in the overall RNA m5C levels during cellular ferroptosis. Furthermore, through immunofluorescence co-localization and RIP techniques, we found that the propensity of methylase NSUN5 to associate with SLC7A11 mRNA, thereby enhancing its protein translation and conferring resistance against ferroptosis. CONCLUSION: RNA methylation is involved in the process of ACLF-associated ferroptosis, and NDD can inhibit ACLF-associated ferroptosis by fostering SLC7A11 m5C methylation.

A facile electrochemical biosensor for coronavirus RNA assay with silver deposition.

Coronaviruses are highly infectious and pose a serious threat to human and animal healths. In this work, a facile electrochemical method based on Exonuclease III (Exo III) catalyzed digestion and silver deposition is developed for coronavirus RNA analysis. A magnetic separation procedure is performed to specifically identify target sequence and release single-stranded DNA modified gold nanoparticles (AuNPs). The nanoparticles can thus be immobilized at a screen-printed electrode and catalyze silver deposition for signal readout. This method allows sensitive analysis of PEDV and SARS-CoV-2 RNAs in the concentration range from 1 to 1000 nM with the limits of detection as low as 0.47 nM and 0.17 nM, respectively. Good specificities are demonstrated. Thus, the proposed method may have great potential use in the applications of coronaviruses analysis.

Genome-Scale Analysis of the Structure and Function of RNA Pathways and Networks in Pseudomonas aeruginosa.

In recent years, several genome-wide approaches based on RNA sequencing (RNA-seq) have been developed. These methods allow a comprehensive and dynamic view of the structure and function of the multi-layered RNA pathways and networks. Many of these approaches, including the promising one of single-cell transcriptome analysis, have been successfully applied to Pseudomonas aeruginosa. However, we are only at the beginning because only a few surrounding conditions have been considered. Here, we aim to illustrate the different types of approaches based on RNA-seq that will lead us in the future to a better understanding of the dynamics of RNA biology in P. aeruginosa.

Quantitative Biochemical Analysis of Deadenylase Enzymes Using Fluorescence and Chemiluminescence-Based Assays.

Deadenylase enzymes play a key role in mRNA degradation and RNA processing. In this chapter, we describe two activity assays for the quantitative biochemical analysis of deadenylase enzymes, which can easily be adapted for other nuclease enzymes. The assays use distinct principles of detection, which are based on differential annealing of a probe complementary to the substrate RNA or detection of adenosine monophosphate (AMP). The assays are sensitive, flexible, and can be used in low-throughput tube-based formats and 96-well or 384-well plate formats. The assays rely on plate reader detection and can be carried out using manual pipetting or robotic liquid handling equipment. In addition to two activity assays, we describe differential scanning fluorimetry (thermal shift assay) as a complementary assay that allows the direct characterization of ligand binding to deadenylase enzymes. The assays can be useful for the characterization of deadenylase variants and are particularly suitable for the discovery and development of small-molecule inhibitors of deadenylase enzymes.