In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific tRNA arginine species for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein to recognize particular arginine tRNAs destined for m3C modification. DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in the
Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in
- PAR ID:
- 10468560
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- EMBO reports
- Volume:
- 24
- Issue:
- 10
- ISSN:
- 1469-221X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract DALRD3 gene that impairs m3C formation in human patients exhibiting developmental delay and early-onset epileptic encephalopathy. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification and suggest a crucial biological role for DALRD3-dependent tRNA modification in proper neurological development. -
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SUMMARY The function of transfer RNAs (tRNAs) depends on enzymes that cleave primary transcript ends, add a 3′ CCA tail, introduce post‐transcriptional base modifications, and charge (aminoacylate) mature tRNAs with the correct amino acid. Maintaining an available pool of the resulting aminoacylated tRNAs is essential for protein synthesis. High‐throughput sequencing techniques have recently been developed to provide a comprehensive view of aminoacylation state in a tRNA‐specific fashion. However, these methods have never been applied to plants. Here, we treated
Arabidopsis thaliana RNA samples with periodate and then performed tRNA‐seq to distinguish between aminoacylated and uncharged tRNAs. This approach successfully captured every tRNA isodecoder family and detected expression of additional tRNA‐like transcripts. We found that estimated aminoacylation rates and CCA tail integrity were significantly higher on average for organellar (mitochondrial and plastid) tRNAs than for nuclear/cytosolic tRNAs. Reanalysis of previously published human cell line data showed a similar pattern. Base modifications result in nucleotide misincorporations and truncations during reverse transcription, which we quantified and used to test for relationships with aminoacylation levels. We also determined that the Arabidopsis tRNA‐like sequences (t‐elements) that are cleaved from the ends of some mitochondrial messenger RNAs have post‐transcriptionally modified bases and CCA‐tail addition. However, these t‐elements are not aminoacylated, indicating that they are only recognized by a subset of tRNA‐interacting enzymes and do not play a role in translation. Overall, this work provides a characterization of the baseline landscape of plant tRNA aminoacylation rates and demonstrates an approach for investigating environmental and genetic perturbations to plant translation machinery. -
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Abstract Background Semaphorin6A (Sema6A) and its PlexinA2 (PlxnA2) receptor canonically function as repulsive axon guidance cues. To understand downstream signaling mechanisms, we performed a microarray screen and identified the “clutch molecule”
shootin‐1 (shtn‐1) as a transcriptionally repressed target. Shtn‐1 is a key proponent of cell migration and neuronal polarization and must be regulated during nervous system development. The mechanisms of Shtn‐1 regulation and the phenotypic consequences of loss of repression are poorly understood.Results We demonstrate
shtn‐1 overexpression results in impaired migration of the optic vesicles, lack of retinal pigmented epithelium, and pathfinding errors of retinotectal projections. We also observed patterning defects in the peripheral nervous system. Importantly, these phenotypes were rescued by overexpressing PlxnA2.Conclusions We demonstrate a functional role for repression of
shtn‐1 by PlxnA2 in development of the eyes and peripheral nervous system in zebrafish. These results demonstrate that careful regulation ofshtn‐1 is critical for development of the nervous system. -
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Abstract Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A‐ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A‐ending codons are not read by U34‐containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A‐ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158–1166, 2019
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Animal and plant microbial pathogens deploy effector proteins and virulence factors to manipulate host cell innate immunity, often using unconventional secretion routes that are poorly understood. Transfer RNA (tRNA) anticodon modifications occur across taxa, but few biological functions are known. Here, in the devastating blast fungus Magnaporthe oryzae, we find that unconventional protein secretion in living host rice cells depends on tRNA modification and codon usage. Using gene deletions, mass spectrometry and live-cell imaging, we characterized the M. oryzae Uba4-Urm1 sulfur relay system mediating tRNA anticodon wobble uridine 2-thiolation (s2U34), a conserved modification required for efficient decoding of AA-ending cognate codons. In M. oryzae, cytoplasmic effectors like Pwl2 and AVR-Pita are translocated into host cells via an unconventional secretion route; apoplastic effectors like Bas4 are secreted by the conventional ER-Golgi pathway. Loss of U34 thiolation abolished PWL2 and AVR-PITA (but not BAS4) mRNA translation in host cells. Paromomycin treatment, which increases near-cognate tRNA acceptance, restored Pwl2 and AVR-Pita production in U34 thiolation-deficient mutant strains. Synonymous AA- to ¬¬AG-ending codon changes remediated PWL2 mRNA translation in uba4; in UBA4+, expressing recoded PWL2 resulted in Pwl2 super-secretion that destabilized the microbe-host cell interface. Thus, wobble U34 tRNA thiolation and codon usage tune pathogen unconventional protein secretion in host cells.more » « less
