RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which enabled us to identify similar modifications in P. aeruginosa. Our analysis revealed a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli. The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which play roles in determining virulence and pathogenicity.

Circadian RNA expression is essential to ultimately regulate a plethora of downstream rhythmic biochemical, physiological, and behavioral processes. Both transcriptional and posttranscriptional mechanisms are considered important to drive rhythmic RNA expression; however, the extent to which each regulatory process contributes to the rhythmic RNA expression remains controversial. To systematically address this, we monitored RNA dynamics using metabolic RNA labeling technology during a circadian cycle in mouse fibroblasts. We find that rhythmic RNA synthesis is the primary contributor of 24-h RNA rhythms, while rhythmic degradation is more important for 12-h RNA rhythms. These rhythms were predominantly regulated by Bmal1 and/or the core clock mechanism, and the interplay between rhythmic synthesis and degradation has a significant impact in shaping rhythmic RNA expression patterns. Interestingly, core clock RNAs are regulated by multiple rhythmic processes and have the highest amplitude of synthesis and degradation, presumably critical to sustain robust rhythmicity of cell-autonomous circadian rhythms. Our study yields invaluable insights into the temporal dynamics of both 24- and 12-h RNA rhythms in mouse fibroblasts.

OBJECTIVE: The glucocorticoid receptor (GR) is a well-studied, ligand-activated transcription factor and a common target of anti-inflammatory treatments. Recently, several studies have drawn attention the effects of binding of GR to RNA rather than DNA and the potential implications of this activity for GR function. The objective of our study was to further characterize the relationship between GR function and RNA binding by measuring changes in the glucocorticoid-driven transcriptome in the presence of a GR mutant that exhibited reduced RNA affinity.
METHODS: GR was activated in three cell lines containing GR constructs (GR-HaloTag). One of the cell lines contained a wild-type GR-HaloTag. Another contained GR-HaloTag with a mutation that reduced RNA affinity and slightly reduced DNA affinity. The third cell line contained GR-HaloTag with a mutation that only slightly reduced DNA affinity. All three cell lines were treated with dexamethasone, a GR agonist. RNA-seq samples were collected every hour for 3 h. Moreover, transcriptome quantification was accomplished via labeling of RNAs transcribed in the final hour of dexamethasone treatment using 4-thiouridine. These labeled RNAs were then purified and sequenced. This data set is the first of its kind for GR and contains valuable insights into the function of RNA binding by GR.

Circadian RNA expression is essential to ultimately regulate a plethora of downstream rhythmic biochemical, physiological, and behavioral processes. Both transcriptional and post-transcriptional mechanisms are considered important to drive rhythmic RNA expression, however, the extent to which each regulatory process contributes to the rhythmic RNA expression remains controversial. To systematically address this, we monitored RNA dynamics using metabolic RNA labeling technology during a circadian cycle in mouse fibroblasts. We find that rhythmic RNA synthesis is the primary contributor of 24 hr RNA rhythms, while rhythmic degradation is more important for 12 hr RNA rhythms. These rhythms were predominantly regulated by Bmal1 and/or the core clock mechanism, and interplay between rhythmic synthesis and degradation has a significant impact in shaping rhythmic RNA expression patterns. Interestingly, core clock RNAs are regulated by multiple rhythmic processes and have the highest amplitude of synthesis and degradation, presumably critical to sustain robust rhythmicity of cell-autonomous circadian rhythms. Our study yields invaluable insights into the temporal dynamics of both 24 hr and 12 hr RNA rhythms in mouse fibroblasts.

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggest the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase. In this review, I focus on the functions and structures of these tRNA modification enzymes and the modified nucleosides they produce. Biochemical, biophysical and structural studies of tRNA 4-thiouridine synthetase, tRNA methyltransferases and tRNA deaminase have established the concept that the THUMP domain captures the 3\'-end of RNA (in the case of tRNA, the CCA-terminus). However, in some cases, this concept is not simply applicable given the modification patterns observed in tRNA. Furthermore, THUMP-related proteins are involved in the maturation of other RNAs as well as tRNA. Moreover, the modified nucleosides, which are produced by the THUMP-related tRNA modification enzymes, are involved in numerous biological phenomena, and the defects of genes for human THUMP-related proteins are implicated in genetic diseases. In this review, these biological phenomena are also introduced.

4-Thiouridine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s4U in tRNA are ongoing in the tRNA modification field. s4U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s4U is essential for progress in the fields of RNA technologies and tRNA modification. Here, we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s4U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation; however, addition of a denaturation step improves the limit of detection. Thermus thermophilus tRNAs, which abundantly contain 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s4U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from a T. thermophilus tRNA 4-thiouridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s4U detection system is that MTSEA biotin-XX reacts only with s4U and not with other sulfur-containing modified nucleosides such as s2U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s4U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s4U detection system can also be used for the analysis of s4U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs in S. cerevisiae cells.

The control of mRNA stability is fundamental to gene regulation, and a deeper understanding of this post-transcriptional regulatory step can provide key insights into gene function. Measuring mRNA half-lives directly, however, is challenging. The most common strategies for evaluating mRNA stability and decay involve blocking general transcription and then measuring the decline in mRNA levels over time. The downside of these approaches, however, is that they severely impact cell function and viability, indirectly perturbing gene expression. Here, we describe Roadblock-qPCR, a simple method for measuring mRNA decay kinetics in living cells that is both economical and quick. Cells are first incubated with the nucleoside analog 4-thiouridine (4sU), which is readily incorporated into nascent mRNAs during transcription. RNA is then extracted and treated with N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that selectively modifies 4sU, before proceeding to cDNA synthesis. Because the NEM-modified 4sU creates a chemical \"roadblock\" that interferes with reverse transcription, this treatment ultimately results in the depletion of the nascent 4sU-containing transcripts from the cDNA pool. As such, the decay rate of the non-4sU-labeled pre-existing mRNAs can be monitored by quantitative PCR (qPCR). In combination with spike-in standards, this approach can be used to efficiently and accurately measure the half-lives of endogenous mRNAs with a wide range of stabilities, while avoiding the artifacts of transcription shutoff strategies. © 2022 Wiley Periodicals LLC. Basic Protocol: Roadblock-qPCR Support Protocol: Synthesis of spike-in mRNA.