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1.
Nucleic Acids Res ; 48(19): 11068-11082, 2020 11 04.
Article in English | MEDLINE | ID: mdl-33035335

ABSTRACT

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.


Subject(s)
Archaeal Proteins , Archaeoglobus fulgidus/enzymology , RNA, Archaeal/metabolism , RNA, Transfer/metabolism , tRNA Methyltransferases , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Protein Conformation , Protein Processing, Post-Translational , tRNA Methyltransferases/chemistry , tRNA Methyltransferases/metabolism
2.
Nucleic Acids Res ; 47(15): 7719-7733, 2019 09 05.
Article in English | MEDLINE | ID: mdl-31328227

ABSTRACT

N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.


Subject(s)
Adenosine/chemistry , Gene Expression Regulation, Neoplastic , Methyltransferases/chemistry , RNA, Messenger/chemistry , RNA, Ribosomal, 18S/chemistry , Adenosine/genetics , Adenosine/metabolism , Base Sequence , Binding Sites , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Cell Line, Tumor , Crystallography, X-Ray , Gene Deletion , HCT116 Cells , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Stability , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Signal Transduction , Substrate Specificity
3.
Nucleic Acids Res ; 46(16): 8483-8499, 2018 09 19.
Article in English | MEDLINE | ID: mdl-30010922

ABSTRACT

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.


Subject(s)
Archaeal Proteins/physiology , Bacterial Proteins/physiology , Haloferax volcanii/enzymology , RNA Processing, Post-Transcriptional , tRNA Methyltransferases/physiology , Bacterial Proteins/genetics , Crystallography, X-Ray , Datasets as Topic , Enzyme Activation , Eukaryotic Cells/enzymology , Evolution, Molecular , Holoenzymes/physiology , Immunoprecipitation , Mass Spectrometry , Methylation , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Mapping , Proteomics , Recombinant Proteins/metabolism , Sequence Alignment , Species Specificity , tRNA Methyltransferases/deficiency , tRNA Methyltransferases/genetics
4.
Biomolecules ; 7(1)2017 01 27.
Article in English | MEDLINE | ID: mdl-28134793

ABSTRACT

Post-transcriptional and post-translational modifications are very important for the control and optimal efficiency of messenger RNA (mRNA) translation. Among these, methylation is the most widespread modification, as it is found in all domains of life. These methyl groups can be grafted either on nucleic acids (transfer RNA (tRNA), ribosomal RNA (rRNA), mRNA, etc.) or on protein translation factors. This review focuses on Trm112, a small protein interacting with and activating at least four different eukaryotic methyltransferase (MTase) enzymes modifying factors involved in translation. The Trm112-Trm9 and Trm112-Trm11 complexes modify tRNAs, while the Trm112-Mtq2 complex targets translation termination factor eRF1, which is a tRNA mimic. The last complex formed between Trm112 and Bud23 proteins modifies 18S rRNA and participates in the 40S biogenesis pathway. In this review, we present the functions of these eukaryotic Trm112-MTase complexes, the molecular bases responsible for complex formation and substrate recognition, as well as their implications in human diseases. Moreover, as Trm112 orthologs are found in bacterial and archaeal genomes, the conservation of this Trm112 network beyond eukaryotic organisms is also discussed.


Subject(s)
Eukaryotic Cells/enzymology , Methyltransferases/metabolism , Protein Biosynthesis , Amino Acid Sequence , Methyltransferases/chemistry , Models, Molecular , Prokaryotic Cells/enzymology
5.
Nucleic Acids Res ; 43(22): 10989-1002, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26438534

ABSTRACT

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm(5)U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.


Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , tRNA Methyltransferases/chemistry , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Saccharomyces cerevisiae Proteins/metabolism , Yarrowia/enzymology , tRNA Methyltransferases/metabolism
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