Biology subjects

Dolce, L. G.

Publications and source records attributed to Dolce, L. G..

3 recordsLinked to original sources

bioRxiv2023

Structural basis of tRNA recognition by the m3C-RNA-methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity 1. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances 2,3. However, our understanding of the substrate selection and catalysis mode of the m3C RNA methyltransferases is currently still lacking. Here, we report the cryo-electron microscopy structure of the m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We reveal how METTL6 and SerRS jointly coordinate the long variable arm of tRNASer in their interface. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, based on the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA binding mode for m3C RNA methyltransferases including METTL2 and METTL8, suggesting that these mammalian paralogues use similar ways to engage their respective tRNA substrates and co-factors.

molecular biology↗

bioRxiv2023

Structural basis for guide RNA selection by the RESC1-RESC2 complex

Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs. Key findingsO_LIThe kinetoplastid mitochondrial RNA editing factors RESC1 and RESC2 resemble a group of capping enzymes that are only found in protozoans, fungi and viruses. C_LIO_LIRESC1 and RESC2 lack the typical catalytic residues, and only RESC2 can bind a triphosphate-nucleoside. C_LIO_LIWe propose that the RESC1-RESC2 dimer selects guide RNAs based on their 5-triphosphate and serves as a protective 5-end binding complex. C_LI

molecular biology↗

bioRxiv2022

Sequence-independent substrate selection by the eukaryotic wobble base deaminase ADAT2/3 involves multiple protein domains and distortion of the tRNA anticodon loop

The essential deamination of adenosine A34 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 first 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. A novel gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.

molecular biology↗