Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

Mitochondrial RNA modification-based signature to predict prognosis of lower grade glioma: a multi-omics exploration and verification study.

Mitochondrial RNA modification (MRM) plays a crucial role in regulating the expression of key mitochondrial genes and promoting tumor metastasis. Despite its significance, comprehensive studies on MRM in lower grade gliomas (LGGs) remain unknown. Single-cell RNA-seq data (GSE89567) was used to evaluate the distribution functional status, and correlation of MRM-related genes in different cell types of LGG microenvironment. We developed an MRM scoring system by selecting potential MRM-related genes using LASSO regression analysis and the Random Survival Forest algorithm, based on multiple bulk RNA-seq datasets from TCGA, CGGA, GSE16011, and E-MTAB-3892. Analysis was performed on prognostic and immunological features, signaling pathways, metabolism, somatic mutations and copy number variations (CNVs), treatment responses, and forecasting of potential small-molecule agents. A total of 35 MRM-related genes were selected from the literature. Differential expression analysis of 1120 normal brain tissues and 529 LGGs revealed that 22 and 10 genes were upregulated and downregulated, respectively. Most genes were associated with prognosis of LGG. METLL8, METLL2A, TRMT112, and METTL2B were extensively expressed in all cell types and different cell cycle of each cell type. Almost all cell types had clusters related to mitochondrial RNA processing, ribosome biogenesis, or oxidative phosphorylation. Cell-cell communication and Pearson correlation analyses indicated that MRM may promoting the development of microenvironment beneficial to malignant progression via modulating NCMA signaling pathway and ICP expression. A total of 11 and 9 MRM-related genes were observed by LASSO and the RSF algorithm, respectively, and finally 6 MRM-related genes were used to establish MRM scoring system (TRMT2B, TRMT11, METTL6, METTL8, TRMT6, and TRUB2). The six MRM-related genes were then validated by qPCR in glioma and normal tissues. MRM score can predict the malignant clinical characteristics, abundance of immune infiltration, gene variation, clinical outcome, the enrichment of signaling pathways and metabolism. In vitro experiments demonstrated that silencing METTL8 significantly curbs glioma cell proliferation and enhances apoptosis. Patients with a high MRM score showed a better response to immunotherapies and small-molecule agents such as arachidonyl trifluoromethyl ketone, MS.275, AH.6809, tacrolimus, and TTNPB. These novel insights into the biological impacts of MRM within the glioma microenvironment underscore its potential as a target for developing precise therapies, including immunotherapeutic approaches.

Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

Identifying Individual Pseudouridine (Ψ) Sites Across Transcripts from HIV-1 Infected Cells.

The identification of RNA modifications at single nucleotide resolution has become an emerging area of interest within biology and specifically among virologists seeking to ascertain how this untapped area of RNA regulation may be altered or hijacked upon viral infection. Herein, we describe a straightforward biochemical approach modified from two original published Ψ mapping protocols, BID-seq and PRAISE, to specifically identify pseudouridine modifications on mRNA transcripts from an HIV-1 infected T cell line. This protocol could readily be adapted for other viral infected cell types and additionally for populations of purified virions from infected cells.

RNA m5C methylation modification: a potential therapeutic target for SARS-CoV-2-associated myocarditis.

The Corona Virus Disease (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has quickly spread worldwide and resulted in significant morbidity and mortality. Although most infections are mild, some patients can also develop severe and fatal myocarditis. In eukaryotic RNAs, 5-methylcytosine (m5C) is a common kind of post-transcriptional modification, which is involved in regulating various biological processes (such as RNA export, translation, and stability maintenance). With the rapid development of m5C modification detection technology, studies related to viral m5C modification are ever-increasing. These studies have revealed that m5C modification plays an important role in various stages of viral replication, including transcription and translation. According to recent studies, m5C methylation modification can regulate SARS-CoV-2 infection by modulating innate immune signaling pathways. However, the specific role of m5C modification in SARS-CoV-2-induced myocarditis remains unclear. Therefore, this review aims to provide insights into the molecular mechanisms of m5C methylation in SARS-CoV-2 infection. Moreover, the regulatory role of NSUN2 in viral infection and host innate immune response was also highlighted. This review may provide new directions for developing therapeutic strategies for SARS-CoV-2-associated myocarditis.

Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

Post-transcriptional (re)programming of B lymphocyte development: From bench to bedside?

Hematopoiesis, a process which generates blood and immune cells, changes significantly during mammalian development. Definitive hematopoiesis is marked by the emergence of long-term hematopoietic stem cells (HSCs). Here, we will focus on the post-transcriptional differences between fetal liver (FL) and adult bone marrow (ABM) HSCs. It remains unclear how or why exactly FL HSCs transition to ABM HSCs, but we aim to leverage their differences to revive an old idea: in utero HSC transplantation. Unexpectedly, the expression of certain RNA-binding proteins (RBPs) play an important role in HSC specification, and can be employed to convert or reprogram adult HSCs back to a fetal-like state. Among other features, FL HSCs have a broad differentiation capacity that includes the ability to regenerate both conventional B and T cells, as well as innate-like or unconventional lymphocytes such as B-1a and marginal zone B (MzB) cells. This chapter will focus on RNA binding proteins, namely LIN28B and IGF2BP3, that are expressed during fetal life and how they promote B-1a cell development. Furthermore, this chapter considers a potential clinical application of synthetic co-expression of LIN28B and IGF2BP3 in HSCs.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

The molecular machinery for maturation of primary mtDNA transcripts.

Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.

Decoding the ribosome's hidden language: rRNA modifications as key players in cancer dynamics and targeted therapies.

Ribosomal RNA (rRNA) modifications, essential components of ribosome structure and function, significantly impact cellular proteomics and cancer biology. These chemical modifications transcend structural roles, critically shaping ribosome functionality and influencing cellular protein profiles. In this review, the mechanisms by which rRNA modifications regulate both rRNA functions and broader cellular physiological processes are critically discussed. Importantly, by altering the translational output, rRNA modifications can shift the cellular equilibrium towards oncogenesis, thus playing a key role in cancer development and progression. Moreover, a special focus is placed on the functions of mitochondrial rRNA modifications and their aberrant expression in cancer, an area with profound implications yet largely uncharted. Dysregulation in these modifications can lead to metabolic dysfunction and apoptosis resistance, hallmark traits of cancer cells. Furthermore, the current challenges and future perspectives in targeting rRNA modifications are highlighted as a therapeutic approach for cancer treatment. In conclusion, rRNA modifications represent a frontier in cancer research, offering novel insights and therapeutic possibilities. Understanding and harnessing these modifications can pave the way for breakthroughs in cancer treatment, potentially transforming the approach to combating this complex disease.

Transfer learning enables identification of multiple types of RNA modifications using nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

Post-Transcriptional HIV-1 Latency: A Promising Target for Therapy?

Human Immunodeficiency Virus type 1 (HIV-1) latency represents a significant hurdle in finding a cure for HIV-1 infections, despite tireless research efforts. This challenge is partly attributed to the intricate nature of HIV-1 latency, wherein various host and viral factors participate in multiple physiological processes. While substantial progress has been made in discovering therapeutic targets for HIV-1 transcription, targets for the post-transcriptional regulation of HIV-1 infections have received less attention. However, cumulative evidence now suggests the pivotal contribution of post-transcriptional regulation to the viral latency in both in vitro models and infected individuals. In this review, we explore recent insights on post-transcriptional latency in HIV-1 and discuss the potential of its therapeutic targets, illustrating some host factors that restrict HIV-1 at the post-transcriptional level.

MLm5C: A high-precision human RNA 5-methylcytosine sites predictor based on a combination of hybrid machine learning models.

RNA modification serves as a pivotal component in numerous biological processes. Among the prevalent modifications, 5-methylcytosine (m5C) significantly influences mRNA export, translation efficiency and cell differentiation and are also associated with human diseases, including Alzheimer's disease, autoimmune disease, cancer, and cardiovascular diseases. Identification of m5C is critically responsible for understanding the RNA modification mechanisms and the epigenetic regulation of associated diseases. However, the large-scale experimental identification of m5C present significant challenges due to labor intensity and time requirements. Several computational tools, using machine learning, have been developed to supplement experimental methods, but identifying these sites lack accuracy and efficiency. In this study, we introduce a new predictor, MLm5C, for precise prediction of m5C sites using sequence data. Briefly, we evaluated eleven RNA sequence-derived features with four basic machine learning algorithms to generate baseline models. From these 44 models, we ranked them based on their performance and subsequently stacked the Top 20 baseline models as the best model, named MLm5C. The MLm5C outperformed the-state-of-the-art predictors. Notably, the optimization of the sequence length surrounding the modification sites significantly improved the prediction performance. MLm5C is an invaluable tool in accelerating the detection of m5C sites within the human genome, thereby facilitating in the characterization of their roles in post-transcriptional regulation.

Nm-Nano: a machine learning framework for transcriptome-wide single-molecule mapping of 2´-O-methylation (Nm) sites in nanopore direct RNA sequencing datasets.

2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.

Exploring the role of ribosomal RNA modifications in cancer.

Recent advances have highlighted the significant roles of post-transcriptional modifications in rRNA in various cancers. Evidence suggests that dysregulation of rRNA modifications acts as a common denominator in cancer development, with alterations in these modifications conferring competitive advantages to cancer cells. Specifically, rRNA modifications modulate protein synthesis and favor the specialized translation of oncogenic programs, thereby contributing to the formation of a protumorigenic proteome in cancer cells. These findings reveal a novel regulatory layer mediated by changes in the deposition of rRNA chemical modifications. Moreover, inhibition of these modifications in vitro and in preclinical studies demonstrates potential therapeutic applications. The recurrence of altered rRNA modification patterns across different types of cancer underscores their importance in cancer progression, proposing them as potential biomarkers and novel therapeutic targets. This review will highlight the latest insights into how post-transcriptional rRNA modifications contribute to cancer progression and summarize the main developments and ongoing challenges in this research area.

Navigating the landscape of epitranscriptomics and host immunity.

RNA modifications, also termed epitranscriptomic marks, encompass chemical alterations to individual nucleotides, including processes such as methylation and editing. These marks contribute to a wide range of biological processes, many of which are related to host immune system defense. The functions of immune-related RNA modifications can be categorized into three main groups: regulation of immunogenic RNAs, control of genes involved in innate immune response, and facilitation of adaptive immunity. Here, we provide an overview of recent research findings that elucidate the contributions of RNA modifications to each of these processes. We also discuss relevant methods for genome-wide identification of RNA modifications and their immunogenic substrates. Finally, we highlight recent advances in cancer immunotherapies that aim to reduce cancer cell viability by targeting the enzymes responsible for RNA modifications. Our presentation of these dynamic research avenues sets the stage for future investigations in this field.

A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3' processing.

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.

A role for a Trypanosoma brucei cytosine RNA methyltransferase homolog in ribosomal RNA processing.

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases. Yet, the identity of the enzymes catalyzing modified RNA base addition and the functions of the enzymes and modifications remain unclear. Homology searches indicate the presence of numerous T. brucei cytosine RNA methyltransferase homologs. One such homolog, TbNop2 was studied in detail. TbNop2 contains the six highly conserved motifs found in cytosine RNA methyltransferases and is evolutionarily related to the Nop2 protein family required for rRNA modification and processing. RNAi experiments targeting TbNop2 resulted in reduced levels of TbNop2 RNA and protein, and a cessation of parasite growth. Next generation sequencing of bisulfite-treated RNA (BS-seq) detected the presence of two methylation sites in the large rRNA; yet TbNop2 RNAi did not result in a significant reduction of methylation. However, TbNop2 RNAi resulted in the retention of 28S internal transcribed spacer RNAs, indicating a role for TbNop2 in rRNA processing.