Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Transcriptomics and proteomics reveal distinct biology for lymph node metastases and tumour deposits in colorectal cancer.

Both lymph node metastases (LNMs) and tumour deposits (TDs) are included in colorectal cancer (CRC) staging, although knowledge regarding their biological background is lacking. This study aimed to compare the biology of these prognostic features, which is essential for a better understanding of their role in CRC spread. Spatially resolved transcriptomic analysis using digital spatial profiling was performed on TDs and LNMs from 10 CRC patients using 1,388 RNA targets, for the tumour cells and tumour microenvironment. Shotgun proteomics identified 5,578 proteins in 12 different patients. Differences in RNA and protein expression were analysed, and spatial deconvolution was performed. Image-based consensus molecular subtype (imCMS) analysis was performed on all TDs and LNMs included in the study. Transcriptome and proteome profiles identified distinct clusters for TDs and LNMs in both the tumour and tumour microenvironment segment, with upregulation of matrix remodelling, cell adhesion/motility, and epithelial-mesenchymal transition (EMT) in TDs (all p < 0.05). Spatial deconvolution showed a significantly increased abundance of fibroblasts, macrophages, and regulatory T-cells (p < 0.05) in TDs. Consistent with a higher fibroblast and EMT component, imCMS classified 62% of TDs as poor prognosis subtype CMS4 compared to 36% of LNMs (p < 0.05). Compared to LNMs, TDs have a more invasive state involving a distinct tumour microenvironment and upregulation of EMT, which are reflected in a more frequent histological classification of TDs as CMS4. These results emphasise the heterogeneity of locoregional spread and the fact that TDs should merit more attention both in future research and during staging. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

STPP-UP: An alternative method for drug target identification using protein thermal stability.

Thermal proteome profiling (TPP) has significantly advanced the field of drug discovery by facilitating proteome-wide identification of drug targets and off-targets. However, TPP has not been widely applied for high-throughput drug screenings, since the method is labor intensive and requires a lot of measurement time on a mass spectrometer. Here, we present Single-tube TPP with Uniform Progression (STPP-UP), which significantly reduces both the amount of required input material and measurement time, while retaining the ability to identify drug targets for compounds of interest. By using incremental heating of a single sample, changes in protein thermal stability across a range of temperatures can be assessed, while alleviating the need to measure multiple samples heated to different temperatures. We demonstrate that STPP-UP is able to identify the direct interactors for anticancer drugs in both human and mice cells. In summary, the STPP-UP methodology represents a useful tool to advance drug discovery and drug repurposing efforts.

Determining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.

In recent years, quantitative mass spectrometry-based interaction proteomics technology has proven very useful in identifying specific DNA-protein interactions using single pull-downs from crude lysates. Here, we applied a SILAC/TMT-based higher-order multiplexing approach to develop an interaction proteomics workflow called Protein-nucleic acid Affinity and Specificity quantification by MAss spectrometry in Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentration range of specific and control DNA baits are performed in SILAC-labeled nuclear extracts. MS1-based quantification to determine specific DNA-protein interactions is then combined with sequential TMT-based quantification of fragmented SILAC peptides, allowing the generation of Hill-like curves and determination of apparent binding affinities. We benchmarked PASMAN using the SP/KLF motif and further applied it to gain insights into two CGCG-containing consensus DNA motifs. These motifs are recognized by two BEN domain-containing proteins, BANP and BEND3, which we find to interact with these motifs with distinct affinities. Finally, we profiled the BEND3 proximal proteome, revealing the NuRD complex as the major BEND3 proximal protein complex in vivo. In summary, PASMAN represents, to our knowledge, the first higher-order multiplexing-based interaction proteomics method that can be used to decipher specific DNA-protein interactions and their apparent affinities in various biological and pathological contexts.

Dynamics and Composition of Small Heat Shock Protein Condensates and Aggregates.

Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.

Quantification of absolute transcription factor binding affinities in the native chromatin context using BANC-seq.

Transcription factor binding across the genome is regulated by DNA sequence and chromatin features. However, it is not yet possible to quantify the impact of chromatin context on transcription factor binding affinities. Here, we report a method called binding affinities to native chromatin by sequencing (BANC-seq) to determine absolute apparent binding affinities of transcription factors to native DNA across the genome. In BANC-seq, a concentration range of a tagged transcription factor is added to isolated nuclei. Concentration-dependent binding is then measured per sample to quantify apparent binding affinities across the genome. BANC-seq adds a quantitative dimension to transcription factor biology, which enables stratification of genomic targets based on transcription factor concentration and prediction of transcription factor binding sites under non-physiological conditions, such as disease-associated overexpression of (onco)genes. Notably, whereas consensus DNA binding motifs for transcription factors are important to establish high-affinity binding sites, these motifs are not always strictly required to generate nanomolar-affinity interactions in the genome.

Gene-by-gene screen of the unknown proteins encoded on Plasmodium falciparum chromosome 3.

Taxon-specific proteins are key determinants defining the biology of all organisms and represent prime drug targets in pathogens. However, lacking comparability with proteins in other lineages makes them particularly difficult to study. In malaria parasites, this is exacerbated by technical limitations. Here, we analyzed the cellular location, essentiality, function, and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nucleus was the most common localization, indicating that it is a hotspot of parasite-specific biology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The mitosis proteins defined a possible orphan complex and a highly diverged complex needed for spindle-kinetochore connection. Structure-function comparisons indicated that the taxon-specific proteins evolved by different mechanisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biology of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

Profiling of plasma extracellular vesicles identifies proteins that strongly associate with patient's global assessment of disease activity in rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation and cartilage/bone damage. Intercellular messengers such as IL-1 and TNF play a crucial role in the pathophysiology of RA but have limited diagnostic and prognostic values. Therefore, we assessed whether the protein content of the recently discovered extracellular vesicles (EVs), which have gained attention in the pathogenesis of RA, correlates with disease activity parameters in RA patients. Methods: We identified and quantified proteins in plasma-derived EVs (pEVs), isolated by size exclusion chromatography from 17 RA patients by mass spectrophotometry (MS). Quantified protein levels were correlated with laboratory and clinical parameters and the patient's own global assessment of their disease activity (PGA-VAS). In a second MS run, the pEV proteins of nine other RA patients were quantified and compared to those from nine healthy controls (HC). Results: No differences were observed in the concentration, size, and protein content of pEVs from RA patients. Proteomics revealed >95% overlapping proteins in RA-pEVs, compared to HC-pEVs (data are available via ProteomeXchange with identifier PXD046058). Remarkably, in both runs, the level of far more RA-pEV proteins correlated positively to PGA-VAS than to either clinical or laboratory parameters. Interestingly, all observed PGA-VAS positively correlated RA-pEV proteins were associated with the actin-cytoskeleton linker proteins, ezrin, and moesin. Conclusion: Our observation suggests that PGA-VAS (loss of vitality) may have a different underlying pathological mechanism in RA, possibly related to enhanced muscle actin-cytoskeleton activity. Furthermore, our study contributes to the growing awareness and evidence that pEVs contain valuable biomarkers for diseases, with added value for RA patients.

SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation-Phosphorylation Cross-talk.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Primary Focal Segmental Glomerulosclerosis Plasmas Increase Lipid Droplet Formation and Perilipin-2 Expression in Human Podocytes.

Many patients with primary focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but were never validated. We aimed to find proteins involved in the mechanism of action of CPF(s) and/or potential biomarkers for the presence of CPF(s). Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s) or healthy and disease controls. Podocyte proteomes were analyzed by LC-MS. Results were validated using flow cytometry, RT-PCR, and immunofluorescence. Podocyte granularity was examined using flow cytometry, electron microscopy imaging, and BODIPY staining. Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, and correlated with the capacity of plasma to induce podocyte granularity, identified as lipid droplet accumulation. Elevated podocyte perilipin-2 was confirmed at protein and mRNA level and was also detected in glomeruli of FSGS patients whose active disease plasmas induced podocyte perilipin-2 and lipid droplets. Our study demonstrates that presumably, CPF-containing plasmas from FSGS patients induce podocyte lipid droplet accumulation and perilipin-2 expression, identifying perilipin-2 as a potential biomarker. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism, which ultimately may result in novel leads for treatment.

Reading ADP-ribosylation signaling using chemical biology and interaction proteomics.

ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.

Identification of Allobaculum mucolyticum as a novel human intestinal mucin degrader.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms.

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

Bifunctional protein PCBD2 operates as a co-factor for hepatocyte nuclear factor 1ß and modulates gene transcription.

Hepatocyte nuclear factor 1ß (HNF1ß) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1ß-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1ß function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1ß interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1ß. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1ß the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1ß to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1ß activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1ß-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1ß-target genes involved in additional processes besides ion transport in the kidney.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan.

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.

EZHIP constrains Polycomb Repressive Complex 2 activity in germ cells.

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.

The interactome of a family of potential methyltransferases in HeLa cells.

Human methytransferase like proteins (METTL) are part of a large protein family characterized by the presence of binding domains for S-adenosyl methionine, a co-substrate for methylation reactions. Despite the fact that members of this protein family were shown or predicted to be DNA, RNA or protein methyltransferases, most METTL proteins are still poorly characterized. Identification of complexes in which these potential enzymes act could help to understand their function(s) and substrate specificities. Here we systematically studied interacting partners of METTL protein family members in HeLa cells using label-free quantitative mass spectrometry. We found that, surprisingly, many of the METTL proteins appear to function outside of stable complexes whereas others including METTL7B, METTL8 and METTL9 have high-confidence interaction partners. Our study is the first systematic and comprehensive overview of the interactome of METTL protein family that can provide a crucial resource for further studies of these potential novel methyltransferases.