Prokaryotic RNA N1-Methyladenosine Erasers Maintain tRNA m1A Modification Levels in .

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, mA tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. MA in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its mA levels in cells.

Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in TrmA and TruB both act as tRNA chaperones, and strains lacking or are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of and in causes a global decrease in aminoacylation and alters other tRNA modifications such as acpU47.

Engineered mischarged transfer RNAs for correcting pathogenic missense mutations.

Missense mutations account for approximately 50% of pathogenic mutations in human genetic diseases, and most lack effective treatments. Gene therapies, gene editing, and RNA therapies, including transfer RNA (tRNA) modalities, are common strategies for genetic disease treatments. However, reported tRNA therapies are for nonsense mutations only.

Single-read tRNA-seq analysis reveals coordination of tRNA modification and aminoacylation and fragmentation.

Transfer RNA (tRNA) utilizes multiple properties of abundance, modification, and aminoacylation in translational regulation. These properties were typically studied one-by-one; however, recent advance in high throughput tRNA sequencing enables their simultaneous assessment in the same sequencing data. How these properties are coordinated at the transcriptome level is an open question.

tRNA abundance, modification and fragmentation in nasopharyngeal swabs as biomarkers for COVID-19 severity.

Emerging and re-emerging respiratory viruses can spread rapidly and cause pandemics as demonstrated by the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The early human immune responses to respiratory viruses are in the nasal cavity and nasopharyngeal regions. Defining biomarkers of disease trajectory at the time of a positive diagnostic test would be an important tool to facilitate decisions such as initiation of antiviral treatment.

Analysis of queuosine and 2-thio tRNA modifications by high throughput sequencing.

Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria.

Structure-activity relationship data and ligand-receptor interactions identify novel agonists consistent with sulfakinin tissue-specific signaling in heart.

Background: The structures and activities of invertebrate sulfakinins that influence gut motility and heart rate are like the vertebrate cholecystokinin (CCK) peptides. Typical of sulfakinin precursors encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2) that bind DSK-R1 and DSK-R2. To explore the role of the nsDSK II N-terminal extension (GGDDQ) in gut we delineated its structure-activity relationship (SAR) and identified novel agonists.

A multiplex platform for small RNA sequencing elucidates multifaceted tRNA stress response and translational regulation.

Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute > 90% of RNA copy numbers in a human cell and perform many essential functions. Popular small RNA-seq strategies limit the insights into coordinated small RNA response to cellular stress. Small RNA-seq also lacks multiplexing capabilities.

Profiling Selective Packaging of Host RNA and Viral RNA Modification in SARS-CoV-2 Viral Preparations.

Viruses package host RNAs in their virions which are associated with a range of functions in the viral life cycle. Previous transcriptomic profiling of host RNA packaging mostly focused on retroviruses. Which host RNAs are packaged in other viruses at the transcriptome level has not been thoroughly examined.

Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling.

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines.

Neuronal Nsun2 deficiency produces tRNA epitranscriptomic alterations and proteomic shifts impacting synaptic signaling and behavior.

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA mC levels, resulting in deficits in expression of 70% of tRNA isodecoders.

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis.

Structural Basis for Teneurin Function in Circuit-Wiring: A Toxin Motif at the Synapse.

Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins.

Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response.

In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses.

Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress.

Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation.

Structure-activity relationships of FMRF-NH2 peptides demonstrate A role for the conserved C terminus and unique N-terminal extension in modulating cardiac contractility.

FMRF-NH2 peptides which contain a conserved, identical C-terminal tetrapeptide but unique N terminus modulate cardiac contractility; yet, little is known about the mechanisms involved in signaling. Here, the structure-activity relationships (SARs) of the Drosophila melanogaster FMRF-NH2 peptides, PDNFMRF-NH2, SDNFMRF-NH2, DPKQDFMRF-NH2, SPKQDFMRF-NH2, and TPAEDFMRF-NH2, which bind FMRFa-R, were investigated. The hypothesis tested was the C-terminal tetrapeptide FMRF-NH2, particularly F1, makes extensive, strong ligand-receptor contacts, yet the unique N terminus influences docking and activity.

Conserved residues in RF-NH₂ receptor models identify predicted contact sites in ligand-receptor binding.

Peptides in the RF-NH2 family are grouped together based on an amidated dipeptide C terminus and signal through G-protein coupled receptors (GPCRs) to influence diverse physiological functions. By determining the mechanisms underlying RF-NH2 signaling targets can be identified to modulate physiological activity; yet, how RF-NH2 peptides interact with GPCRs is relatively unexplored. We predicted conserved residues played a role in Drosophila melanogaster RF-NH2 ligand-receptor interactions.