5-Methoxycarbonylmethyl-2-thiouridine - CAS 20299-15-4

The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.

Urm1 (ubiquitin related modifier 1) is a molecular fossil in the class of ubiquitin-like proteins (UBLs). It encompasses characteristics of classical UBLs, such as ubiquitin or SUMO (small ubiquitin-related modifier), but also of bacterial sulfur-carrier proteins (SCP). Since its main function is to modify tRNA, Urm1 acts in a non-canonical manner. Uba4, the activating enzyme of Urm1, contains two domains: a classical E1-like domain (AD), which activates Urm1, and a rhodanese homology domain (RHD). This sulfurtransferase domain catalyzes the formation of a C-terminal thiocarboxylate on Urm1. Thiocarboxylated Urm1 is the sulfur donor for 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), a chemical nucleotide modification at the wobble position in tRNA. This thio-modification is conserved in all domains of life and optimizes translation. The absence of Urm1 increases stress sensitivity in yeast triggered by defects in protein homeostasis, a hallmark of neurological defects in higher organisms. In contrast, elevated levels of tRNA modifying enzymes promote the appearance of certain types of cancer and the formation of metastasis. Here, we summarize recent findings on the unique features that place Urm1 at the intersection of UBL and SCP and make Urm1 an excellent model for studying the evolution of protein conjugation and sulfur-carrier systems.

There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection of RNA modifications requires working with nucleosides in bulk samples. Nanopore detection technology uses a single-molecule approach that has the potential to detect tRNA modifications. To evaluate the feasibility of this approach, we have performed all-atom molecular dynamics (MD) simulation studies of a five-layered graphene nanopore by localizing canonical and modified uridine nucleosides. We found that in a 1 M KCl solution with applied positive and negative biases not exceeding 2 V, nanopores can distinguish U from 5-carbonylmethyluridine (cm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) based on changes in the resistance of the nanopore. Specifically, we observed that in nanopores with dimensions less than 3 nm diameter, a localized mcm5Um and mcm5U modifications could be clearly distinguished from the canonical uridine, while the other modifications showed a modest yet detectable decrease in their respective nanopore conductance. We have compared the results between nanopores of various sizes to aid in the design, optimization, and fabrication of graphene nanopores devices for tRNA modification detection.