Eukaryotic elongation factor 1A (eEF1A) is an essential and highly conserved protein involved in diverse cellular processes, including translation, cytoskeleton organisation, nuclear export, and ...proteasomal degradation. Recently, nine novel and site-specific methyltransferases were discovered that target eEF1A, five in yeast and four in human, making it the eukaryotic protein with the highest number of independent methyltransferases. Some of these methyltransferases show striking evolutionary conservation. Yet, they come from diverse methyltransferase families, indicating they confer competitive advantage through independent origins. As might be expected, the first functional studies of specific methylation sites found them to have distinct effects, notably on eEF1A-related processes of translation and tRNA aminoacylation. Further functional studies of sites will likely reveal other unique roles for this interesting modification.
eEF1A is a highly modified protein involved in many different cellular processes, including translation, actin cytoskeleton organisation, proteasomal degradation, and nuclear export.
It has been known for decades that eEF1A contains many methylated residues; despite this, the responsible methyltransferases were not known.
Recent studies have uncovered nine novel methyltransferases, four in human and five in yeast, that target specific eEF1A residues. Some of these enzymes are conserved from yeast to human.
eEF1A methyltransferases come from diverse methyltransferase families, including two families that were only discovered recently.
Knockout studies of three human eEF1A methyltransferases suggest a role for methylation in eEF1A-related processes, such as translation and tRNA aminoacylation.
Recent studies strongly indicate that aberrations in the control of gene expression might contribute to the initiation and progression of Alzheimer's disease (AD). In particular, alternative splicing ...has been suggested to play a role in spontaneous cases of AD. Previous transcriptome profiling of AD models and patient samples using microarrays delivered conflicting results. This study provides, for the first time, transcriptomic analysis for distinct regions of the AD brain using RNA-Seq next-generation sequencing technology. Illumina RNA-Seq analysis was used to survey transcriptome profiles from total brain, frontal and temporal lobe of healthy and AD post-mortem tissue. We quantified gene expression levels, splicing isoforms and alternative transcript start sites. Gene Ontology term enrichment analysis revealed an overrepresentation of genes associated with a neuron's cytological structure and synapse function in AD brain samples. Analysis of the temporal lobe with the Cufflinks tool revealed that transcriptional isoforms of the apolipoprotein E gene, APOE-001, -002 and -005, are under the control of different promoters in normal and AD brain tissue. We also observed differing expression levels of APOE-001 and -002 splice variants in the AD temporal lobe. Our results indicate that alternative splicing and promoter usage of the APOE gene in AD brain tissue might reflect the progression of neurodegeneration.
The transient elevation of cytosolic free calcium concentration (Ca2+cyt) induced by cold stress is a well‐established phenomenon; however, the underlying mechanism remains elusive. Here, we report ...that the Ca2+‐permeable transporter ANNEXIN1 (AtANN1) mediates cold‐triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold‐induced Ca2+cyt increase and upregulation of the cold‐responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold‐induced Ca2+cyt elevation in the ost1‐3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1‐AtANN1 to cold‐induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.
Synopsis
How cold stress generates and controls transient elevation of calcium in the cytosol of plant cells is ill‐defined. Cold‐activated OST1 kinase promotes freezing tolerance in Arabidopsis by potentiating ANNEXIN1‐dependent calcium signaling.
ANNEXIN1 (AtANN1) mediates the generation of cold‐triggered Ca2+ signals.
Cold‐activated OST1 kinase phosphorylates AtANN1 to enhance its Ca2+ transport activity, thereby amplifying Ca2+ signaling.
OST1‐AtANN1 module regulates the expression of cold responsive CBF and COR genes and plant freezing tolerance.
Cold‐activated OST1 kinase phosphorylates ANNEXIN1 to boost calcium ion uptake and thus promotes expression of COR genes that enhance cold stress responses in Arabidopsis.
RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their ...identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.
Synopsis
A new method uses UV‐crosslinking of RNase E–RNA complexes to map the small RNA–RNA interaction network in bacteria and provide transcriptome‐wide information on small RNA function.
The small RNA–RNA interaction network in bacteria is captured by UV‐crosslinking of RNase E–RNA complexes in vivo.
RNase E binding sites are identified in a transcriptome‐wide manner.
Small RNAs direct target RNA cleavage 3′ of duplexed seed sequences.
The enterohaemorrhagic E. coli‐specific sRNA, Esr41, confers colicin 1A resistance.
A new method using UV‐crosslinking maps the small RNA–RNA interaction network in bacteria and provides transcriptome‐wide information on small RNA function.
The field of proteomics is increasingly concerned with the diversity and functional relevance of protein modifications. Differential ion mobility spectrometry (DMS) is emerging as a tool to detect ...and quantify additional peptide and protein species that are difficult to analyse with conventional instrumental methods. In this review, recent advances in DMS are discussed, with a focus on the different types of DMS instruments now available to researchers in proteomics. Furthermore, the combination of DMS with mass spectrometry (MS) for increased proteome coverage and the targeted analysis of modification patterns on single proteins is highlighted. Frontier areas of research, such as the analysis of intact proteoforms, and future directions for the implementation of DMS in proteomics are also discussed.
DMS can be used to rapidly separate ions prior to MS analysis.
Compared to drift tube-based IMS, ion separation by DMS is more orthogonal to MS.
The implementation of DMS in MS workflows has improved the coverage and quantification of proteomes, including the large-scale analysis of PTMs.
High-resolution DMS can separate isobaric peptides that are otherwise challenging to separate by use of conventional chromatography methods, including peptides that differ by the position of a single methyl group.
DMS combined with other gas-phase separation methods in tandem ion mobility can provide an alternative to LC, for exceptionally high-throughput proteomic analyses.
Defining all sites for a post-translational modification in the cell, and identifying their upstream modifying enzymes, is essential for a complete understanding of a modification's function. ...However, the complete mapping of a modification in the proteome and definition of its associated enzyme-substrate network is rarely achieved. Here, we present the protein methylation network for
Through a formal process of defining and quantifying all potential sources of incompleteness, for both the methylation sites in the proteome and also protein methyltransferases, we prove that this protein methylation network is now near-complete. It contains 33 methylated proteins and 28 methyltransferases, comprising 44 enzyme-substrate relationships, and a predicted further three enzymes. While the precise molecular function of most methylation sites is unknown, and it remains possible that other sites and enzymes remain undiscovered, the completeness of this protein modification network is unprecedented and allows us to holistically explore the role and evolution of protein methylation in the eukaryotic cell. We show that while no single protein methylation event is essential in yeast, the vast majority of methylated proteins are themselves essential, being primarily involved in the core cellular processes of transcription, RNA processing, and translation. This suggests that protein methylation in lower eukaryotes exists to fine-tune proteins whose sequences are evolutionarily constrained, providing an improvement in the efficiency of their cognate processes. The approach described here, for the construction and evaluation of post-translational modification networks and their constituent enzymes and substrates, defines a formal process of utility for other post-translational modifications.
Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. Here, we comprehensively review the function and regulation of the histone methylation network in ...the budding yeast and model eukaryote, Saccharomyces cerevisiae. First, we outline the lysine methylation sites that are found on histone proteins in yeast (H3K4me1/2/3, H3K36me1/2/3, H3K79me1/2/3, and H4K5/8/12me1) and discuss their biological and cellular roles. Next, we detail the reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes that are known to control histone lysine methylation in budding yeast cells. Specifically, we illustrate the domain architecture of the methylation enzymes and highlight the structural features that are required for their respective functions and molecular interactions. Finally, we discuss the prevalence of post-translational modifications on yeast histone methylation enzymes and how phosphorylation, acetylation, and ubiquitination in particular are emerging as key regulators of enzyme function. We note that it will be possible to completely connect the histone methylation network to the cell’s signaling system, given that all methylation sites and cognate enzymes are known, most phosphosites on the enzymes are known, and the mapping of kinases to phosphosites is tractable owing to the modest set of protein kinases in yeast. Moving forward, we expect that the rich variety of post-translational modifications that decorates the histone methylation machinery will explain many of the unresolved questions surrounding the function and dynamics of this intricate epigenetic network.
The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ...ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential cross-linking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture cross-linking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine–lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein–protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how cross-linking MS will be an essential tool for understanding complex phenotypes.
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•Hpm1p-mediated histidine methylation of Rpl3p localized to H243 by mass spectrometry.•Novel extraribosomal functional phenotypes found on loss of Hpm1p.•Large-scale in vivo quantitative and comparative crosslinking analysis performed.•Changes detected in structures and interactions, independent of protein abundance.•Phenotypes could be explained or predicted through crosslinking analysis.
The yeast histidine methyltransferase Hpm1p targets a ribosomal substrate but has many unexplained extraribosomal phenotypes. To understand these, we used protein expression analysis and quantitative cross-linking mass spectrometry to compare WT and Δhpm1 cells. This revealed a role of Hpm1p in metabolism, mitochondria, and membranes. Cross-linking mass spectrometry detected changes in protein structures and interactions and was a powerful means of understanding phenotype. Insights were different to those gained from expression analysis, making these techniques of great potential.
In this review, we describe the attributes of histone H3 mutants identified in cancer. H3 mutants were first identified in genes encoding H3.3, in pediatric high-grade glioma, and subsequently in ...chondrosarcomas and giant cell tumors of bone (GCTB) in adolescents. The most heavily studied are the lysine to methionine mutants K27M and K36M, which perturb the target site for specific lysine methyltransferases and dominantly perturb methylation of corresponding lysines in other histone H3 proteins. We discuss recent progress in defining the consequences of these mutations on chromatin, including a newly emerging view of the central importance of the disruption of H3K36 modification in many distinct K to M histone mutant cancers. We also review new work exploring the role of H3.3 G34 mutants identified in pediatric glioma and GCTB. G34 is not itself post-translationally modified, but G34 mutation impinges on the modification of H3K36. Here, we ask if G34R mutation generates a new site for methylation on the histone tail. Finally, we consider evidence indicating that histone mutations might be more widespread in cancer than previously thought, and if the perceived bias towards mutation of H3.3 is real or reflects the biology of tumors in which the histone mutants were first identified.
The methylation of eukaryotic proteins has been proposed to be widespread, but this has not been conclusively shown to date. In this study, we examined 36,854 previously generated peptide mass ...spectra from 2,607 Saccharomyces cerevisiae proteins for the presence of arginine and lysine methylation. This was done using the FindMod tool and 5 filters that took advantage of the high number of replicate analysis per protein and the presence of overlapping peptides.
A total of 83 high-confidence lysine and arginine methylation sites were found in 66 proteins. Motif analysis revealed many methylated sites were associated with MK, RGG/RXG/RGX or WXXXR motifs. Functionally, methylated proteins were significantly enriched for protein translation, ribosomal biogenesis and assembly and organellar organisation and were predominantly found in the cytoplasm and ribosome. Intriguingly, methylated proteins were seen to have significantly longer half-life than proteins for which no methylation was found. Some 43% of methylated lysine sites were predicted to be amenable to ubiquitination, suggesting methyl-lysine might block the action of ubiquitin ligase.
This study suggests protein methylation to be quite widespread, albeit associated with specific functions. Large-scale tandem mass spectroscopy analyses will help to further confirm the modifications reported here.
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