Trypanosomes as a magnifying glass for cell and molecular biology.

The African trypanosome, Trypanosoma brucei, has developed into a flexible and robust experimental model for molecular and cellular parasitology, allowing us to better combat these and related parasites that cause worldwide suffering. Diminishing case numbers, due to efficient public health efforts, and recent development of new drug treatments have reduced the need for continued study of T. brucei in a disease context.

The tRNA methyltransferase TrmB is critical for stress responses and pulmonary infection.

As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the mG tRNA methyltransferase TrmB is crucial for a top-priority pathogen, , to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation.

tRNA has an unusually short half-life in .

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes.

Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3.

The essential deamination of adenosine A to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core.

Genomic and transcriptomic profiling of phoenix colonies.

Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media.

METTLing in the right place: METTL8 is a mitochondrial tRNA-specific methyltransferase.

Schöller et al. (2021) discovered that METTL8, thought of as an mRNA modifier, is a tRNA-specific mitochondrial enzyme important for mitochondrial translation and function. Paradoxically, increased expression of METTL8 is associated with high respiratory rates in pancreatic cancers.

The importance of RNA modifications: From cells to muscle physiology.

Naturally occurring post-transcriptional chemical modifications serve critical roles in impacting RNA structure and function. More directly, modifications may affect RNA stability, intracellular transport, translational efficiency, and fidelity. The combination of effects caused by modifications are ultimately linked to gene expression regulation at a genome-wide scale.

Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis.

Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems.

From canonical to modified nucleotides: balancing translation and metabolism.

Every type of nucleic acid in cells may undergo some kind of post-replicative or post-transcriptional chemical modification. Recent evidence has highlighted their importance in biology and their chemical complexity. In the following pages, we will describe new discoveries of modifications, with a focus on tRNA and mRNA.

RNA-Binding Proteins and Their Targets in Trypanosoma brucei: Single Nucleotide Resolution Using iCLIP and iCLAP.

RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs.

The general mRNA exporters Mex67 and Mtr2 play distinct roles in nuclear export of tRNAs in Trypanosoma brucei.

Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm.

How the intracellular partitioning of tRNA and tRNA modification enzymes affects mitochondrial function.

Organisms have evolved different strategies to seclude certain molecules to specific locations of the cell. This is most pronounced in eukaryotes with their extensive intracellular membrane systems. Intracellular compartmentalization is particularly critical in genome containing organelles, which because of their bacterial evolutionary ancestry still maintain protein-synthesis machinery that resembles more their evolutionary origin than the extant eukaryotic cell they once joined as an endosymbiont.

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei.

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei.

Binding synergy as an essential step for tRNA editing and modification enzyme codependence in .

Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, , a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of mC and mU at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly codependent in Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction.

Retrograde nuclear transport from the cytoplasm is required for tRNA maturation in T. brucei.

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve.

The role of intracellular compartmentalization on tRNA processing and modification.

A signature of most eukaryotic cells is the presence of intricate membrane systems. Intracellular organization presumably evolved to provide order, and add layers for regulation of intracellular processes; compartmentalization also forcibly led to the appearance of sophisticated transport systems. With nucleus-encoded tRNAs, it led to the uncoupling of tRNA synthesis from many of the maturation steps it undergoes.

The Evolution of Substrate Specificity by tRNA Modification Enzymes.

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability.

Editing and methylation at a single site by functionally interdependent activities.

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro.

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs.

Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3΄-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life.