Identification of Protein Isoforms Using Reference Databases Built from Long and Short Read RNA-Sequencing.

Alternative splicing can lead to distinct protein isoforms. These can have different functions in specific cells and tissues or in different developmental stages. In this study, we explored whether transcripts assembled from long read, nanopore-based, direct RNA-sequencing (RNA-seq) could improve the identification of protein isoforms in human K562 cells. By comparing with Illumina-based short read RNA-seq, we showed that a large proportion of Ensembl transcripts (5949/14,326) and genes expressing alternatively spliced transcripts (486/2981) identified with long direct reads were missed by short paired-end reads. By co-analyzing proteomic and transcriptomic data, we also showed that some peptides (826/35,976), proteins (262/3215), and protein isoforms arising from distinct transcript variants (574/1212) identified with isoform-specific peptides via custom long-read-based databases were missed in Illumina-derived databases. Finally, we generated unequivocal peptide evidence for a set of protein isoforms and showed that long read, direct RNA-seq allows the discovery of novel protein isoforms not already in reference databases or custom databases built from short read RNA-seq data. Our analysis highlights the benefits of long read RNA-seq data in the generation of reference databases to increase tandem mass spectrometry (MS/MS) identification of protein isoforms.

Systematic investigation of PRMT6 substrate recognition reveals broad specificity with a preference for an RG motif or basic and bulky residues.

Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here, we systematically characterise the substrate recognition motif of PRMT6, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure their effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. We then quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 tolerates essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 also has preference for glycine, but only in the position immediately following the target arginine. This indicates that PRMT6 recognises the RG motif rather than the RGG motif. We further confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity and recognition of RG rather than RGG are distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6. DATABASES: Panorama Public (https://panoramaweb.org/PRMT6motif.url); ProteomeXchange (PXD016711).

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

Cross-linking Mass Spectrometry Analysis of the Yeast Nucleus Reveals Extensive Protein-Protein Interactions Not Detected by Systematic Two-Hybrid or Affinity Purification-Mass Spectrometry.

Saccharomyces cerevisiae has the most comprehensively characterized protein-protein interaction network, or interactome, of any eukaryote. This has predominantly been generated through multiple, systematic studies of protein-protein interactions by two-hybrid techniques and of affinity-purified protein complexes. A pressing question is to understand how large-scale cross-linking mass spectrometry (XL-MS) can confirm and extend this interactome. Here, intact yeast nuclei were subject to cross-linking with disuccinimidyl sulfoxide (DSSO) and analyzed using hybrid MS2-MS3 methods. XlinkX identified a total of 2,052 unique residue pair cross-links at 1% FDR. Intraprotein cross-links were found to provide extensive structural constraint data, with almost all intralinks that mapped to known structures and slightly fewer of those mapping to homology models being within 30 Å. Intralinks provided structural information for a further 366 proteins. A method for optimizing interprotein cross-link score cut-offs was developed, through use of extensive known yeast interactions. Its application led to a high confidence, yeast nuclear interactome. Strikingly, almost half of the interactions were not previously detected by two-hybrid or AP-MS techniques. Multiple lines of evidence existed for many such interactions, whether through literature or ortholog interaction data, through multiple unique interlinks between proteins, and/or through replicates. We conclude that XL-MS is a powerful means to measure interactions, that complements two-hybrid and affinity-purification techniques.

Histone H3 Mutations: An Updated View of Their Role in Chromatin Deregulation and Cancer.

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.

Knockout of the Hmt1p Arginine Methyltransferase in Saccharomyces cerevisiae Leads to the Dysregulation of Phosphate-associated Genes and Processes.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1Δ, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3 Analysis of the hmt1Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1Δ cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

MT-MAMS: Protein Methyltransferase Motif Analysis by Mass Spectrometry.

Protein methyltransferases often recognize their substrates through linear sequence motifs. The determination of these motifs is critical to understand methyltransferase mechanism, function, and drug targeting. Here we describe MT-MAMS (methyltransferase motif analysis by mass spectrometry), a quantitative approach to characterize methyltransferase substrate recognition motifs. In MT-MAMS, peptide sets are synthesized which contain all amino acid substitutions at single positions within a template sequence. These are then incubated with the methyltransferase of interest in the presence of deuterated S-adenosyl methionine (D3-AdoMet). The use of this heavy methyl donor gives unique mass shifts to methylated peptides, allowing their unambiguous quantification by mass spectrometry. The stoichiometry of methylation resulting from each substitution is then derived, and finally the methyltransferase substrate recognition motif is generated. We validated MT-MAMS by application to lysine methyltransferase G9a, generating the substrate recognition motif (TKRN)-(A > RS > G)-(R ≫ K)-K-(STRCKMAQHG)-Φ; this is highly similar to that previously determined by peptide arrays. We then determined the recognition motif of yeast lysine elongation factor methyltransferase 1 (Efm1) to be (Y > FW)-K-^P-G-G-Φ. This is a new type of lysine methyltransferase recognition motif that only contains noncharged residues, excluding the target lysine. We further determined recognition motifs of major yeast and human arginine methyltransferases Hmt1 and PRMT1, revealing them to be ^(DE)-^(DE)-R-(G ≫ A)-(GN > RAW)-(FYW > ILKHM) and ^(DE)-^(DE)-R-(G ≫ N)-(GR > ANK)-(K > YHMFILW), respectively. These motifs expand significantly on the canonical RGG recognition motif and include the negative specificity of these enzymes, a feature unique to MT-MAMS. Finally, we show that MT-MAMS can be used to generate insights into the processivity of protein methyltransferases.

The activity of a yeast Family 16 methyltransferase, Efm2, is affected by a conserved tryptophan and its N-terminal region.

The Family 16 methyltransferases are a group of eukaryotic nonhistone protein methyltransferases. Sixteen of these have recently been described in yeast and human, but little is known about their sequence and structural features. Here we investigate one of these methyltransferases, Saccharomyces cerevisiae elongation factor methyltransferase 2 (Efm2), by site-directed mutagenesis and truncation. We show that an active site-associated tryptophan, invariant in Family 16 methyltransferases and at position 222 in Efm2, is important for methyltransferase activity. A second highly conserved tryptophan, at position 318 in Efm2, is likely involved in S-adenosyl methionine binding but is of lesser consequence for catalysis. By truncation analysis, we show that the N-terminal 50-200 amino acids of Efm2 are critical for its methyltransferase activity. As N-terminal regions are variable among Family 16 methyltransferases, this suggests a possible role in determining substrate specificity. This is consistent with recently solved structures that show the core of Family 16 methyltransferases to be near-identical but the N termini to be structurally quite different. Finally, we show that Efm2 can exist as an oligomer but that its N terminus is not necessary for oligomerisation to occur.