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Unknown,Transcriptomics,Genomics,Proteomics

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NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation

ABSTRACT: Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilization of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. HEK293 cell lines expressing His-FLAG-tagged NSUN3 or the His-FLAG tag alone were crosslinked using UV or treated with 5-azacytidine and analysed by CRAC

ORGANISM(S): Homo sapiens

SUBMITTER: Markus Bohnsack

PROVIDER: E-GEOD-84664 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation

Project description:Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilization of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.

2016-08-09 | GSE84664 | GEO

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs

Project description:Mitochondria contain a specific translation machinery for the synthesis of respiratory chain components encoded on the mitochondrial genome. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial genome and, similar to their cytoplasmic counterparts, are modified at various positions. Here, we find that the RNA methyltransferase METTL8, is a mitochondrial protein that facilitates m3C methylation at position C32 of mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knock out cells show reduced and over expressing cells enhanced respiratory chain activity. In pancreatic cancer, METTL8 levels are high, which correlates with patient survival. Indeed, METTL8 up regulation stimulates respiratory chain activity in these cells. Ribosome occupancy analysis using ribosome profiling revealed ribosome stalling on mt-tRNASer(UCN) and mt-tRNAThr codons and mass spectrometry analysis of native ribosomal subcomplexes unraveled reduced respiratory chain incorporation of the mitochondria encoded proteins ND6 and ND1. A well-balanced translation of mt-tRNASer(UCN) and mt-tRNAThr codons through METTL8-mediated C32 methylation might therefore provide optimal respiratory chain compositions and function.

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FOXK2 ChIP-seq Illumina

Project description:Chromatin immunoprecipitation of FOXK2 (tagged with Flag and His tags) in U2OS cells detected by Illumina sequencing

2014-07-23 | E-MTAB-2204 | biostudies-arrayexpress

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts [PAR-CLIP]

Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with HEK293 cell lines stably expressing HIS/FLAG/HA-tagged ALKBH5, C17orf85, C22orf28, CAPRIN1, and ZC3H7B. We used 4-thiouridine (4SU) to enhance the crosslink and generated two PAR-CLIP cDNA libraries per protein.

2012-06-01 | E-GEOD-38201 | biostudies-arrayexpress

FOXK2 ChIP-seq

Project description:Chromatin immunoprecipitation of FOXK2 (tagged with Flag and His tags) in U2OS cells detected by SOLiD sequencing. ***Correction March 2014: The sample “FOXK2_Dox_treated” has been renamed, it was originally named “FOXK2_rep2”. A new sample “FOXK2_rep2” has been added, with new files. It has come to our attention that one of the FOXK2 ChIP-seq replicates 'FOXK2_rep2' that we used in our paper recent paper (Ji, Z., Donaldson, I.J., Liu, J., Hayes, A., Zeef, L.A.H. and Sharrocks, A.D. (2012) The forkhead transcription factor FOXK2 promotes AP-1-mediated transcriptional regulation. Mol. Cell. Biol. 32, 385-398. doi:10.1128/MCB.05504-11) was incorrect. The replicate was actually treated with doxorubicin prior to ChIP-seq analysis resulting in the loss of many FOXK2 binding events.***

2011-01-20 | E-MTAB-565 | biostudies-arrayexpress

FOXK2 ChIP-chip

Project description:Chromatin immunoprecipitation of FOXK2 (tagged with Flag and His tags) in U2OS cells detected by Affymetrix human promoter array 1.0R.

2011-11-03 | E-MEXP-3116 | biostudies-arrayexpress

SLAM-seq for K562 endogenous mRNA decay

Project description:mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.

2019-05-01 | GSE126522 | GEO

Codon usage influences the local rate of translation elongation to regulate co-translational protein folding

Project description:Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics. The role of codon usage in regulating translation, however, is not clear and has been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the use of preferred codons enhances the rate of translation elongation, whereas non-optimal codons slow translation. In addition, codon usage regulates ribosome traffic on the mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with substrate mRNAs manipulated to increase signal over background noise. We further show that codon usage plays an important role in regulating protein function by affecting co-translational protein folding. Together, these results resolve a long-standing fundamental question and demonstrate the importance of codon usage on protein folding.

2015-07-18 | GSE71032 | GEO

endogenous mRNA decay in 293t, hela, RPE cell line

Project description:mRNA endogenous decay profiles approach in 293T, HeLa and RPE cells. mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.

2019-05-01 | GSE126520 | GEO

N-terminal amino acid identification using targeted mass spectrometry

Project description:Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA.exp lacking all UAG stop codons was created, freeing this ‘amber’ stop codon for other purposes. An engineered ‘amber initiator’ that activates translation at UAG codons is available, but little is known about amino acid it assigns to the amber start codon. Here, we combine for the first time the amber initiator tRNA in C321.ΔA.exp and measure its cellular effects. Using, targeted proteomics, we found that the amber initiator tRNA initiates proteins exclusively with methionine.

2019-03-15 | PXD012390 | panorama

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