Binding synergy as an essential step for tRNA editing and modification enzyme co-dependence in Trypanosoma brucei.

RNA. 2017 Oct 17. pii: rna.062893.117. doi: 10.1261/rna.062893.117. [Epub ahead of print]

McKenney KM1, Rubio MAT1, Alfonzo JD2.

Author information

Abstract

Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, Trypanosoma brucei, a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of m3C and m3U at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly co-dependent in T. brucei Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction. Although these enzymes interact directly with each other, tRNA binding assays using enzyme variants mutated in critical binding and catalytic site indicate that the observed binding synergy stems from contributions from tRNA-binding domains distal to their active sites. These results provide a rationale for the known interactions of these proteins, while also speaking to the modulation of substrate specificity between seemingly unrelated enzymes. This information should be of value in furthering our understanding of how tRNA modification enzymes act together to regulate gene expression at the posttranscriptional level and provide a basis for the interdependence of such activities.

Published by Cold Spring Harbor Laboratory Press for the RNA Society.

KEYWORDS: 

RNA Editing; T. brucei; modification; tRNA

PMID:

 

29042505

 

DOI:

 

10.1261/rna.062893.117

Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei

RNA Biol. 2017 Sep 13:0. doi: 10.1080/15476286.2017.1377878. [Epub ahead of print]

Kessler AC1,2, Kulkarni SS3, Paulines MJ4, Rubio MAT1,2, Limbach PA4, Paris Z3, Alfonzo JD1,5,2.

Author information

Abstract

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote T. brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

KEYWORDS: 

intron; queuosine; retrograde; splicing; tRNA; transport

PMID:

 

28901827

 

DOI:

 

10.1080/15476286.2017.1377878

The role of intracellular compartmentalization on tRNA processing and modification

RNA Biol. 2017 Aug 29:1-13. doi: 10.1080/15476286.2017.1371402. [Epub ahead of print]

Kessler AC1,2, Silveira d'Almeida G1,2, Alfonzo JD1,2,3.

Author information

Abstract

A signature of most eukaryotic cells is the presence of intricate membrane systems. Intracellular organization presumably evolved to provide order, and add layers for regulation of intracellular processes; compartmentalization also forcibly led to the appearance of sophisticated transport systems. With nucleus-encoded tRNAs, it led to the uncoupling of tRNA synthesis from many of the maturation steps it undergoes. It is now clear that tRNAs are actively transported across intracellular membranes and at any point, in any compartment, they can be post-transcriptionally modified; modification enzymes themselves may localize to any of the genome-containing compartments. In the following pages, we describe a number of well-known examples of how intracellular compartmentalization of tRNA processing and modification activities impact the function and fate of tRNAs. We raise the possibility that rates of intracellular transport may influence the level of modification and as such increase the diversity of differentially modified tRNAs in cells.

KEYWORDS: 

Maturation; modification; nuclear export; retrograde transport; tRNA splicing

PMID:

 

28850002

 

DOI:

 

10.1080/15476286.2017.1371402

The Evolution of Substrate Specificity by tRNA Modification Enzymes

Enzymes. 2017;41:51-88. doi: 10.1016/bs.enz.2017.03.002. Epub 2017 Apr 26.

McKenney KM1, Rubio MAT1, Alfonzo JD2.

Author information

Abstract

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability. Whichever the case, the overall importance of modifications is highlighted by both their evolutionary conservation and the fact that organisms use a substantial portion of their genomes to encode modification enzymes, far exceeding what is needed for the de novo synthesis of the canonical nucleotides themselves [2]. Although some modifications occur at exactly the same nucleotide position in tRNAs from the three domains of life, many can be found at various positions in a particular tRNA and their location may vary between and within different tRNAs. With this wild array of chemical diversity and substrate specificities, one of the big challenges in the tRNA modification field has been to better understand at a molecular level the modes of substrate recognition by the different modification enzymes; in this realm RNA binding rests at the heart of the problem. This chapter will focus on several examples of modification enzymes where their mode of RNA binding is well understood; from these, we will try to draw general conclusions and highlight growing themes that may be applicable to the RNA modification field at large.

© 2017 Elsevier Inc. All rights reserved.

KEYWORDS: 

Editing; Methylation; Pseudouridine; RNA binding

PMID: 

28601226DOI:10.1016/bs.enz.2017.03.002

Nucleic Acids Res. 2017 Feb 28;45(4):2124-2136. doi: 10.1093/nar/gkw1120.

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs.

Kang BI1, Miyauchi K1, Matuszewski M2, D'Almeida GS3, Rubio MAT3, Alfonzo JD3, Inoue K1, Sakaguchi Y1, Suzuki T1, Sochacka E2, Suzuki T1.

Author information

1

Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan.

2

Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Lodz 90-924, Poland.

3

Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.

Abstract

Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3΄-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life. t6A and its derivatives have pleiotropic functions in protein synthesis including aminoacylation, decoding and translocation. We previously discovered a cyclic form of t6A (ct6A) as a chemically labile derivative of t6A in tRNAs from bacteria, fungi, plants and protists. Here, we report 2-methylthio cyclic t6A (ms2ct6A), a novel derivative of ct6A found in tRNAs from Bacillus subtilis, plants and Trypanosoma brucei. In B. subtilis and T. brucei, ms2ct6A disappeared and remained to be ms2t6A and ct6A by depletion of tcdA and mtaB homologs, respectively, demonstrating that TcdA and MtaB are responsible for biogenesis of ms2ct6A.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

PMID:

 

27913733

 

PMCID:

 

PMC5389704

 

DOI:

 

10.1093/nar/gkw1120

Nature. 2017 Feb 22;542(7642):494-497. doi: 10.1038/nature21396.

Editing and methylation at a single site by functionally interdependent activities.

Rubio MA1, Gaston KW1,2, McKenney KM1, Fleming IM1, Paris Z1,3, Limbach PA2, Alfonzo JD1.

Author information

1

Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.

2

Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, USA.

3

Institute of Parasitology, Biology Centre and Faculty of Sciences, University of South Bohemia, 37005 České Budějovice (Budweis), Czech Republic.

Abstract

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.

Comment in

• Molecular biology: RNA editing packs a one-two punch. [Nature. 2017]

PMID:

 

28230119

 

DOI:

 

10.1038/nature21396

Methods. 2016 Sep 1;107:1-2. doi: 10.1016/j.ymeth.2016.08.007.

Post-transcriptional RNA modification methods.

Alfonzo JD.

PMID:

 

27600834

 

DOI:

 

10.1016/j.ymeth.2016.08.007

RNA. 2016 Aug;22(8):1190-9. doi: 10.1261/rna.056242.116. Epub 2016 Jun 9. 

PMID:

 

27284166

The essential function of the Trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr.

Lopes RR1, Silveira Gde O1, Eitler R2, Vidal RS2, Kessler A3, Hinger S3, Paris Z4, Alfonzo JD3, Polycarpo C1.

Author information

1

Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Caixa Postal 68041, Brazil Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Caixa Postal 68041, Brazil.

2

Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Caixa Postal 68041, Brazil.

3

Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.

4

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, 37005 České Budějovice (Budweis), Czech Republic.

Abstract

Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function. 

© 2016 Lopes et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications

Life 2016, 6(1), 13; doi:10.3390/life6010013 (registering DOI)

Received: 11 January 2016 / Revised: 27 February 2016 / Accepted: 7 March 2016 / Published: 14 March 2016  (This article belongs to the Special Issue Evolution of tRNA) 

Review
Katherine M. McKenney^1,2  and Juan D. Alfonzo^1,2,3,*

¹ The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA ² The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA ³ Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA 

Abstract

All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes.

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A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei.

Sci Rep 2016 Feb 18;6:21438. doi: 10.1038/srep21438.

Fleming IM¹, Paris Z¹, Gaston KW², Balakrishnan R^1,2, Fredrick K^1,3, Rubio MA¹, Alfonzo JD^1,3. 

 

Abstract

Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells. 

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