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Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ZNHIT3 (zinc finger HIT type containing protein 3) is an evolutionarily conserved protein required for ribosome biogenesis by mediating the assembly of small nucleolar RNAs (snoRNAs) of class C/D into ribonucleoprotein complexes (snoRNPs). Missense mutations in the gene encoding ZNHIT3 protein have been previously reported to cause PEHO syndrome, a severe neurodevelopmental disorder typically presenting after birth. We discuss here the case of two fetuses from a single family who presented with isolated hydrops during the early second trimester of pregnancy, resulting in intrauterine demise. Autopsy revealed no associated malformation. Through whole-genome quartet analysis, we identified two novel variants within the ZNHIT3 gene, both inherited from healthy parents and occurring as compound heterozygotes in both fetuses. The c.40T>C p.Cys14Arg variant originated from the father, while the c.251_254delAAGA variant was of maternal origin. Analysis of the variants in human cell culture models reveals that both variants reduce cell growth, albeit to different extents, and impact the protein's stability and function in distinct ways. The c.251_254delAAGA results in production of a stable form of ZNHIT3 that lacks a domain required for mediating snoRNP biogenesis, whereas the c.40T>C p.Cys14Arg variation behaves similarly to the previously described PEHO-associated ZNHIT3 variants that destabilize the protein. Interestingly, both variations lead to a marked decrease in specific box C/D snoRNA levels, reduced rRNA levels and cellular translation. Analysis of rRNA methylation pattern in fetus samples reveals distinct sites of hypo 2'-O-methylation. RNA-seq analysis of undifferentiated and differentiated SHSY5Y cells transfected with the ZNHIT3 variants reveals differential expression of a set of genes, many of which are associated with developmental processes and RNA binding compared to cells expressing wild-type ZNHIT3. In summary, this work extends the phenotype of PEHO syndrome to include antenatal manifestations and describe the molecular defects induced by two novel ZNHIT3 variants., Competing Interests: Competing interests The authors report no competing interests.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives., Competing Interests: Competing interests statement:The authors declare no competing interest.

Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m 1 A mRNA methylation is highly discussed due to methodological differences. However, one single m 1 A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m 1 A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid β (Aβ). Aβ mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m 1 A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m 1 A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m 1 A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aβ-induced mitochondrial dysfunction., (© 2024. The Author(s).)

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens- Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana . Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. Bartonella quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Quaiyum, Sun, Marchand, Sun, Reed, Motorin, Dedon, Minnick and de Crécy-Lagard.)

In the past decade tRNA sequencing (tRNA-seq) has attracted considerable attention as an important tool for the development of novel approaches to quantify highly modified tRNA species and to propel tRNA research aimed at understanding the cellular physiology and disease and development of tRNA-based therapeutics. Many methods are available to quantify tRNA abundance while accounting for modifications and tRNA charging/acylation. Advances in both library preparation methods and bioinformatic workflows have enabled developments in next-generation sequencing (NGS) workflows. Other approaches forgo NGS applications in favor of hybridization-based approaches. In this review we provide a brief comparative overview of various tRNA quantification approaches, focusing on the advantages and disadvantages of these methods, which together facilitate reliable tRNA quantification., Competing Interests: Declaration of interests We have no conflicts of interest to declare., (Published by Elsevier Ltd.)

Among the many analytical methods applied to RNA modifications, a particularly pronounced surge has occurred in the past decade in the field of modification mapping. The occurrence of modifications such as m 6 A in mRNA, albeit known since the 1980s, became amenable to transcriptome-wide analyses through the advent of next-generation sequencing techniques in a rather sudden manner. The term "mapping" here refers to detection of RNA modifications in a sequence context, which has a dramatic impact on the interpretation of biological functions. As a consequence, an impressive number of mapping techniques were published, most in the perspective of what now has become known as "epitranscriptomics". While more and more different modifications were reported to occur in mRNA, conflicting reports and controversial results pointed to a number of technical and theoretical problems rooted in analytics, statistics, and reagents. Rather than finding the proverbial needle in a haystack, the tasks were to determine how many needles of what color in what size of a haystack one was looking at.As the authors of this Account, we think it important to outline the limitations of different mapping methods since many life scientists freshly entering the field confuse the accuracy and precision of modification mapping with that of normal sequencing, which already features numerous caveats by itself. Indeed, we propose here to qualify a specific mapping method by the size of the transcriptome that can be meaningfully analyzed with it.We here focus on high throughput sequencing by Illumina technology, referred to as RNA-Seq. We noted with interest the development of methods for modification detection by other high throughput sequencing platforms that act directly on RNA, e.g., PacBio SMRT and nanopore sequencing, but those are not considered here.In contrast to approaches relying on direct RNA sequencing, current Illumina RNA-Seq protocols require prior conversion of RNA into DNA. This conversion relies on reverse transcription (RT) to create cDNA; thereafter, the cDNA undergoes a sequencing-by-synthesis type of analysis. Thus, a particular behavior of RNA modified nucleotides during the RT-step is a prerequisite for their detection (and quantification) by deep sequencing, and RT properties have great influence on the detection efficiency and reliability. Moreover, the RT-step requires annealing of a synthetic primer, a prerequisite with a crucial impact on library preparation. Thus, all RNA-Seq protocols must feature steps for the introduction of primers, primer landing sites, or adapters on both the RNA 3'- and 5'-ends.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress., (© 2023. The Author(s).)

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35 S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33 , but not SNORA22 , reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N 7 -methylguanosine (m 7 G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m 7 G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m 7 G tRNA methylation in cancer cell translation control and tumour biology., (© 2023. The Author(s).)