Isotope-based analysis of modified tRNA nucleosides correlates modification density with translational efficiency.
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ABSTRACT:
SUBMITTER: Brandmayr C
PROVIDER: S-EPMC3533783 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted. Therefore, thermophiles must possess strategies to stabilize tRNA structures. To this end, several thermophile-specific modified nucleosides in tRNA have been identified. Other factors such as RNA-binding proteins and polyamines contribute to the stability of tRNA at high temperatures. Thermus thermophilus, which is an extreme-thermophilic eubacterium, can adapt its protein synthesis system in response to temperature changes via the network of modified nucleosides in tRNA and tRNA modification enzymes. Notably, tRNA modification enzymes from thermophiles are very stable. Therefore, they have been utilized for biochemical and structural studies. In the future, thermostable tRNA modification enzymes may be useful as biotechnology tools and may be utilized for medical science.
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Project description:RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA's cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.
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Project description:The existence of SpoU-TrmD (SPOUT) RNA methyltransferase superfamily was first predicted by bioinformatics. SpoU is the previous name of TrmH, which catalyzes the 2'-Omethylation of ribose of G18 in tRNA; TrmD catalyzes the formation of N1-methylguanosine at position 37 in tRNA. Although SpoU (TrmH) and TrmD were originally considered to be unrelated, the bioinformatics study suggested that they might share a common evolution origin and form a single superfamily. The common feature of SPOUT RNA methyltransferases is the formation of a deep trefoil knot in the catalytic domain. In the past decade, the SPOUT RNA methyltransferase superfamily has grown; furthermore, knowledge concerning the functions of their modified nucleosides in tRNA has also increased. Some enzymes are potential targets in the design of antibacterial drugs. In humans, defects in some genes may be related to carcinogenesis. In this review, recent findings on the tRNA methyltransferases with a SPOUT fold and their methylated nucleosides in tRNA, including classification of tRNA methyltransferases with a SPOUT fold; knot structures, domain arrangements, subunit structures and reaction mechanisms; tRNA recognition mechanisms, and functions of modified nucleosides synthesized by this superfamily, are summarized. Lastly, the future perspective for studies on tRNA modification enzymes are considered.
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Project description:We identified a novel N1-methyladenosine (m1A) modification in tRNALys with distinct effects on the synthesis and stabilities of the 13 mitochondrial proteins. Moreover, we observed regulated RNA modifications of mitochondrial tRNAs for the control of mitochondrial gene expression. Collectively, our data provide unprecedented insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.
2018-10-29 | GSE106601 | GEO
Project description:BackgroundRNA modifications have been proven to play fundamental roles in regulating cellular biology process. Recently, maladjusted N7-methylguanosine (m7G) modification and its modifiers METTL1/WDR4 have been confirmed an oncogene role in multiple cancers. However, the functions and molecular mechanisms of METTL1/WDR4 in acute myeloid leukemia (AML) remain to be determined.MethodsMETTL1/WDR4 expression levels were quantified using qRT-PCR, western blot analysis on AML clinical samples, and bioinformatics analysis on publicly available AML datasets. CCK-8 assays and cell count assays were performed to determine cell proliferation. Flow cytometry assays were conducted to assess cell cycle and apoptosis rates. Multiple techniques were used for mechanism studies in vitro assays, such as northern blotting, liquid chromatography-coupled mass spectrometry (LC-MS/MS), tRNA stability analysis, transcriptome sequencing, small non-coding RNA sequencing, quantitative proteomics, and protein synthesis measurements.ResultsMETTL1/WDR4 are significantly elevated in AML patients and associated with poor prognosis. METTL1 knockdown resulted in reduced cell proliferation and increased apoptosis in AML cells. Mechanically, METTL1 knockdown leads to significant decrease of m7G modification abundance on tRNA, which further destabilizes tRNAs and facilitates the biogenesis of tsRNAs in AML cells. In addition, profiling of nascent proteins revealed that METTL1 knockdown and transfection of total tRNAs that were isolated from METTL1 knockdown AML cells decreased global translation efficiency in AML cells.ConclusionsTaken together, our study demonstrates the important role of METTL1/WDR4 in AML leukaemogenesis, which provides a promising target candidate for AML therapy.
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Project description:Because binding of RNAP to misplaced sites could compromise the efficiency of transcription, natural selection for the optimization of gene expression should regulate the distribution of DNA motifs capable of RNAP-binding across the genome. Here we analyze the distribution of the -10 promoter motifs that bind the sigma(70) subunit of RNAP in 42 bacterial genomes. We show that selection on these motifs operates across the genome, maintaining an over-representation of -10 motifs in regulatory sequences while eliminating them from the nonfunctional and, in most cases, from the protein coding regions. In some genomes, however, -10 sites are over-represented in the coding sequences; these sites could induce pauses effecting regulatory roles throughout the length of a transcriptional unit. For nonfunctional sequences, the extent of motif under-representation varies across genomes in a manner that broadly correlates with the number of tRNA genes, a good indicator of translational speed and growth rate. This suggests that minimizing the time invested in gene transcription is an important selective pressure against spurious binding. However, selection against spurious binding is detectable in the reduced genomes of host-restricted bacteria that grow at slow rates, indicating that components of efficiency other than speed may also be important. Minimizing the number of RNAP molecules per cell required for transcription, and the corresponding energetic expense, may be most relevant in slow growers. These results indicate that genome-level properties affecting the efficiency of transcription and translation can respond in an integrated manner to optimize gene expression. The detection of selection against promoter motifs in nonfunctional regions also confirms previous results indicating that no sequence may evolve free of selective constraints, at least in the relatively small and unstructured genomes of bacteria.
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Project description:Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading.
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Project description:The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The importance of t6A in decoding has been studied for the past 40 years, but the cellular role has remained unclear. In this work, we examined the genome-wide effect to translation during the absence of t6A. In accordance with the previous experiments using single gene reporters, absence of t6A led to a increase of upstream non-AUG initiation of translation and increased translation ambiguities. Surprisingly, absence of t6A did not lead to a global and cataclysmic frame-shifting, but instead occurred only in a few genes. Restoration of protein homeostasis through the overexpression of Unfolded Protein Response (UPR) factors or the addition of L-homoserine suppressed the slow growth phenotype seen in t6A-defcient strains. 4 samples
2016-02-15 | E-GEOD-72030 | biostudies-arrayexpress
Project description:Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2’-O-methylguanosine, 2’-O-methylcytidine, N4-acetylcytidine and 5-formylcytidine), and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs, Rra1. Additionally, we find that mRNA modification levels change in response to heat shock, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.
2019-06-20 | GSE126405 | GEO
Project description:The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase. In this review, I focus on the functions and structures of these tRNA modification enzymes and the modified nucleosides they produce. Biochemical, biophysical and structural studies of tRNA 4-thiouridine synthetase, tRNA methyltransferases and tRNA deaminase have established the concept that the THUMP domain captures the 3'-end of RNA (in the case of tRNA, the CCA-terminus). However, in some cases, this concept is not simply applicable given the modification patterns observed in tRNA. Furthermore, THUMP-related proteins are involved in the maturation of other RNAs as well as tRNA. Moreover, the modified nucleosides, which are produced by the THUMP-related tRNA modification enzymes, are involved in numerous biological phenomena, and the defects of genes for human THUMP-related proteins are implicated in genetic diseases. In this review, these biological phenomena are also introduced.
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