Multi-Substrate Specificity and the Evolutionary Basis for Interdependence in tRNA Editing and Methylation Enzymes

Dixit, Sameer; Henderson, Jeremy C.; Alfonzo, Juan D.

doi:10.3389/fgene.2019.00104

Cited by 21 publications

(23 citation statements)

References 133 publications

(235 reference statements)

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“…The METTL2-DALRD3 complex presents yet another case of a multi-subunit enzyme catalyzing tRNA modification. The emerging number of multimeric tRNA modification enzymes has been hypothesized to be driven by the need to recognize and modify different substrates while maintaining high specificity 55,56 . In one pertinent example, the activity of S. cerevisiae Trm140p on seryl-tRNA substrates is greatly stimulated by interaction with seryl-tRNA synthetase, which can recognize the full cellular repertoire of seryl-tRNAs even though their anticodons do not have any nucleotides in common 25 .…”

Section: Discussionmentioning

confidence: 99%

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification

Lentini

Alsaif

Faqeih³

et al. 2020

Nat Commun

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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. DALRD3deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in the 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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Section: Discussionmentioning

confidence: 99%

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification

Lentini

Alsaif

Faqeih³

et al. 2020

Nat Commun

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“…Transfer RNAs (tRNAs) play a critical role in the translation of mRNA into proteins. Eukaryotic tRNA genes are transcribed by RNA Pol III and undergo a series of post-transcriptional processing and modification steps prior to reaching their mature and functional state (1)(2)(3)(4)(5)(6). A subset of tRNA genes contain introns that must be removed during tRNA maturation.…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Hayne

Schmidt²,

Matera³

et al. 2019

Preprint

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The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.We thank Drs. Andrew Sikkema and Marcos Morgan as well as members of the Stanley Lab for their critical reading of this manuscript. We would like to acknowledge Robert Petrovich and the NIEHS Protein Expression Facility for assistance with protein expression in HEK293 suspension cultures and for supplying the anti-GFP resin. We also thank Robert Dutcher for his assistance with running SEC-MALS.

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“…The METTL2-DALRD3 complex presents a new case of a multi-subunit enzyme catalyzing tRNA modification. The emerging number of multimeric tRNA modification enzymes has been hypothesized to be driven by the need to recognize and modify different substrates while maintaining high specificity (Dixit et al, 2019;Guy and Phizicky, 2014).…”

Section: Discussionmentioning

confidence: 99%

METTL2 forms a complex with the DALRD3 anticodon-domain binding protein to catalyze formation of 3-methylcytosine in specific arginine tRNA isoacceptors

Lentini

2019

Preprint

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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 arginine tRNAs 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 in order to recognize particular arginine tRNAs destined for m3C modification. Using biochemical reconstitution, we find that METTL2-DALDR3 complexes catalyze m3C formation in vitro that is dependent upon sequence elements specific to certain arginine tRNAs. Notably, DALRD3deficient human cells exhibit nearly complete loss of the m3C modification in arginine tRNAs.These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification.

show abstract

Multi-Substrate Specificity and the Evolutionary Basis for Interdependence in tRNA Editing and Methylation Enzymes

Cited by 21 publications

References 133 publications

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

METTL2 forms a complex with the DALRD3 anticodon-domain binding protein to catalyze formation of 3-methylcytosine in specific arginine tRNA isoacceptors

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