Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis.

Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis in vivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity in vitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs that target growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure are critical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response.

The helicase RIG-I initiates an antiviral immune response after recognition of pathogenic RNA. TRIM25, an E3 ubiquitin ligase, mediates K63-linked ubiquitination of RIG-I, which is crucial for RIG-I downstream signaling and the antiviral innate immune response. The components and mode of the RIG-I-initiated innate signaling remain to be fully understood. Here we identify a novel long noncoding RNA (Lnczc3h7a) that binds to TRIM25 and promotes RIG-I-mediated antiviral innate immune responses. Depletion of Lnczc3h7a impairs RIG-I signaling and the antiviral innate response to RNA viruses in vitro and in vivo. Mechanistically, Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold for stabilization of the RIG-I-TRIM25 complex at the early stage of viral infection. Lnczc3h7a facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and thus promotes downstream signaling transduction. Our findings reveal that host RNAs can enhance the response of innate immune sensors to foreign RNAs, ensuring effective antiviral defense.

Nuclear RNF2 inhibits interferon function by promoting K33-linked STAT1 disassociation from DNA.

Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.

Chemical linguistics: Reading the modified proteome.

In this issue of Molecular Cell, Lin et al. (2021) develop a tri-functional amino acid probe for the discovery and characterization of protein domains that sense or "read" protein post-translational modifications, a chemical tool that can facilitate our understanding of how signaling networks act at the molecular level.

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications3-5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

Affinity selection of double-click triazole libraries for rapid discovery of allosteric modulators for GLP-1 receptor.

The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

Inhalable Stem Cell Exosomes Promote Heart Repair After Myocardial Infarction.

BACKGROUND: Exosome therapy shows potential for cardiac repair after injury. However, intrinsic challenges such as short half-life and lack of clear targets hinder the clinical feasibility. Here, we report a noninvasive and repeatable method for exosome delivery through inhalation after myocardial infarction (MI), which we called stem cell-derived exosome nebulization therapy (SCENT). METHODS: Stem cell-derived exosomes were characterized for size distribution and surface markers. C57BL/6 mice with MI model received exosome inhalation treatment through a nebulizer for 7 consecutive days. Echocardiographies were performed to monitor cardiac function after SCENT, and histological analysis helped with the investigation of myocardial repair. Single-cell RNA sequencing of the whole heart was performed to explore the mechanism of action by SCENT. Last, the feasibility, efficacy, and general safety of SCENT were demonstrated in a swine model of MI, facilitated by 3-dimensional cardiac magnetic resonance imaging. RESULTS: Recruitment of exosomes to the ischemic heart after SCENT was detected by ex vivo IVIS imaging and fluorescence microscopy. In a mouse model of MI, SCENT ameliorated cardiac repair by improving left ventricular function, reducing fibrotic tissue, and promoting cardiomyocyte proliferation. Mechanistic studies using single-cell RNA sequencing of mouse heart after SCENT revealed a downregulation of Cd36 in endothelial cells (ECs). In an EC-Cd36fl/- conditional knockout mouse model, the inhibition of CD36, a fatty acid transporter in ECs, led to a compensatory increase in glucose utilization in the heart and higher ATP generation, which enhanced cardiac contractility. In pigs, cardiac magnetic resonance imaging showed an enhanced ejection fraction (Δ=11.66±5.12%) and fractional shortening (Δ=5.72±2.29%) at day 28 after MI by SCENT treatment compared with controls, along with reduced infarct size and thickened ventricular wall. CONCLUSIONS: In both rodent and swine models, our data proved the feasibility, efficacy, and general safety of SCENT treatment against acute MI injury, laying the groundwork for clinical investigation. Moreover, the EC-Cd36fl/- mouse model provides the first in vivo evidence showing that conditional EC-CD36 knockout can ameliorate cardiac injury. Our study introduces a noninvasive treatment option for heart disease and identifies new potential therapeutic targets.

PIF1 helicase promotes break-induced replication in mammalian cells.

Break-induced replication (BIR) is a specialized homologous-recombination pathway for DNA double-strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP-based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1-dependent BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation-prone AT-rich DNA sequences derived from common fragile sites (CFS-ATs) induce BIR upon replication stress and oncogenic stress, and PCNA-dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC-mediated repair of double-ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease-generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS-ATs. The breast cancer-associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.

High-throughput saturation mutagenesis generates a high-affinity antibody against SARS-CoV-2 variants using protein surface display assay on a human cell.

As new mutations continue to emerge, the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus to evade the human immune system and neutralizing antibodies remains a huge challenge for vaccine development and antibody research. The majority of neutralizing antibodies have reduced or lost activity against SARS-CoV-2 variants. In this study, we reported a novel protein surface display system on a mammalian cell for obtaining a higher-affinity antibody in high-throughput manner. Using a saturation mutagenesis strategy through integrating microarray-based oligonucleotide synthesis and single-cell screening assay, we generated a group of new antibodies against diverse prevalent SARS-CoV-2 variants through high-throughput screening the human antibody REGN10987 within 2 weeks. The affinity of those optimized antibodies to seven prevalent mutants was greatly improved, and the EC50 values were no higher than 5 ng/mL. These results demonstrate the robustness of our screening system in the rapid generation of an antibody with higher affinity against a new SARS-CoV-2 variant, and provides a potential application to other protein molecular interactions.

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition.

Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.

Strong effect-correlated electrochemical CO2 reduction.

Electrochemical CO2 reduction (ECR) holds great potential to alleviate the greenhouse effect and our dependence on fossil fuels by integrating renewable energy for the electrosynthesis of high-value fuels from CO2. However, the high thermodynamic energy barrier, sluggish reaction kinetics, inadequate CO2 conversion rate, poor selectivity for the target product, and rapid electrocatalyst degradation severely limit its further industrial-scale application. Although numerous strategies have been proposed to enhance ECR performances from various perspectives, scattered studies fail to comprehensively elucidate the underlying effect-performance relationships toward ECR. Thus, this review presents a comparative summary and a deep discussion with respect to the effects strongly-correlated with ECR, including intrinsic effects of materials caused by various sizes, shapes, compositions, defects, interfaces, and ligands; structure-induced effects derived from diverse confinements, strains, and fields; electrolyte effects introduced by different solutes, solvents, cations, and anions; and environment effects induced by distinct ionomers, pressures, temperatures, gas impurities, and flow rates, with an emphasis on elaborating how these effects shape ECR electrocatalytic activities and selectivity and the underlying mechanisms. In addition, the challenges and prospects behind different effects resulting from various factors are suggested to inspire more attention towards high-throughput theoretical calculations and in situ/operando techniques to unlock the essence of enhanced ECR performance and realize its ultimate application.

CPA4 as a biomarker promotes the proliferation, migration and metastasis of clear cell renal cell carcinoma cells.

Clear cell renal cell carcinoma (ccRCC) is a commonly occurring and highly aggressive urological malignancy characterized by a significant mortality rate. Current therapeutic options for advanced ccRCC are limited, necessitating the discovery of novel biomarkers and therapeutic targets. Carboxypeptidase A4 (CPA4) is a zinc-containing metallocarboxypeptidase with implications in various cancer types, but its role in ccRCC remains unexplored. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized in order to investigate the differential expression patterns of CPA4. The expression of CPA4 in ccRCC patients was further verified using immunohistochemical (IHC) examination of 24 clinical specimens. A network of protein-protein interactions (PPI) was established, incorporating CPA4 and its genes that were expressed differentially. Functional enrichment analyses were conducted to anticipate the contribution of CPA4 in the development of ccRCC. To validate our earlier study, we conducted real-time PCR and cell functional tests on ccRCC cell lines. Our findings revealed that CPA4 is overexpressed in ccRCC, and the higher the expression of CPA4, the worse the clinical outcomes such as TNM stage, pathological stage, histological grade, etc. Moreover, patients with high CPA4 expression had worse overall survival, disease-specific survival and progress-free interval than patients with low expression. The PPI network analysis highlighted potential interactions contributing to ccRCC progression. Functional enrichment analysis indicated the involvement of CPA4 in the regulation of key pathways associated with ccRCC development. Additionally, immune infiltration analysis suggested a potential link between CPA4 expression and immune response in the tumour microenvironment. Finally, cell functional studies in ccRCC cell lines shed light on the molecular mechanisms underlying the role of CPA4 in promoting ccRCC formation. Overall, our study unveils CPA4 as a promising biomarker with prognostic potential in ccRCC. The identified interactions and pathways provide valuable insights into its implications in ccRCC development and offer a foundation for future research on targeted therapies. Further investigation of CPA4's involvement in immune responses may contribute to the development of immunotherapeutic strategies for ccRCC treatment.

DNA repair protein RAD52 is required for protecting G-quadruplexes in mammalian cells.

G-quadruplex (G4)-forming DNA sequences are abundant in the human genome, and they are hot spots for inducing DNA double-strand breaks (DSBs) and genome instability. The mechanisms involved in protecting G4s and maintaining genome stability have not been fully elucidated. Here, we demonstrated that RAD52 plays an important role in suppressing DSB accumulation at G4s, and RAD52-deficient cells are sensitive to G4-stabilizing compounds. Mechanistically, we showed that RAD52 is required for efficient homologous recombination repair at G4s, likely due to its function in recruiting structure-specific endonuclease XPF to remove G4 structures at DSB ends. We also demonstrated that upon G4 stabilization, endonuclease MUS81 mediates cleavage of stalled replication forks at G4s. The resulting DSBs recruit RAD52 and XPF to G4s for processing DSB ends to facilitate homologous recombination repair. Loss of RAD52 along with G4-resolving helicase FANCJ leads to a significant increase of DSB accumulation before and after treatment with the G4-stabilizing compound pyridostatin, and RAD52 exhibits a synthetic lethal interaction with FANCJ. Collectively, our findings reveal a new role of RAD52 in protecting G4 integrity and provide insights for new cancer treatment strategies.

Antibody toolkit to investigate eEF1A methylation dynamics in mRNA translation elongation.

Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.