Biology subjects

Dong, M.-Q.

Publications and source records attributed to Dong, M.-Q..

At least 19 recordsLinked to original sources

bioRxiv2026

RNabel-A Standalone Software Tool for Annotating Tandem Mass Spectra of Modified Ribonucleic Acids

Ribonucleic acid (RNA) modifications, with over 170 identified types, play diverse roles in cellular processes. The past decade has witnessed surging demand for accurate identification and localization of RNA modifications in both endogenous and synthetic therapeutic RNAs. With accurate spectral annotation for RNA, tandem mass spectrometry (MS/MS) can meet this demand. Here we present RNabel, a user-friendly software tool for in-depth annotation of MS/MS spectra of RNA oligonucleotides. RNabel considers a full set of backbone-cleavage ions (a, b, c, d, a-B, w, x, y, z) in which the ribonucleotide unit could be A, U, C, G, Y (pseudouridine), or I (Inosine). Additionally, RNabel considers 196 modifications on the base, the phosphoribose linkage, the 5' or the 3' terminus, or detachment of a sub-nucleotide fragment as a neutral or charged group. Users can create new components if needed, including ribonucleotides, modifications, neutral or charged groups that could detach from a ribonucleotide. RNabel efficiently processes large datasets in four acceptable formats including .mgf, .raw, .txt from msConvert, and RNabel batch files. Multiple statistical metrics are provided for quality assessment of spectral annotation. To accelerate RNA modification analysis, RNabel is made freely available for Mac and Windows users at https://github.com/songge1111/RNabel/releases. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/733900v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@65af35org.highwire.dtl.DTLVardef@1d1c2d4org.highwire.dtl.DTLVardef@4e04d2org.highwire.dtl.DTLVardef@1eb2a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

Selective activation of IRE-1 safeguards restoration of translation and organismal rejuvenation from adult reproductive diapause

In C. elegans, prolonged food deprivation during early larval development results in aging-like phenotypes that can be fully reversed upon refeeding, but it remains unknown whether and how this ability persists into later life stages. Here we subjected C. elegans of the last larval stage to starvation, driving them into adult reproductive diapause (ARD). During starvation, ARD worms exhibited a wide spectrum of aging-like phenotypes, including transcriptomic reprogramming accompanying cellular and functional declines. These phenotypes are largely restored within one day of refeeding, suggesting a rejuvenation effect. Time-series transcriptomics, proteomics, and follow-up analyses of the refeeding/rejuvenation process uncovered an intricate coordination between: (1) activation of the IRE-1 branch of UPRER (unfolded protein response of endoplasmic reticulum) to induce chaperone expression; (2) quiescence of the PEK-1 branch of UPRER to avoid translation suppression; (3) up-regulation of the translation machinery to boost protein synthesis. IRE-1, together with its downstream effector, XBP-1, play an essential role in boosting protein synthesis, which is required for complete rejuvenation from the ARD state. These findings indicate that coordination between a high protein synthesis activity and a high protein folding capacity is key to refeeding-associated rejuvenation. HighlightsO_LIRefeeding rapidly reverses aging-like phenotypes in adult reproductive diapause C_LIO_LIRefeeding selectively activates the IRE-1 branch of UPRER C_LIO_LIRefeeding restores protein synthesis in an IRE-1 dependent manner C_LIO_LIRejuvenation requires enhanced translation activity and protein folding capacity C_LI

cell biology↗

bioRxiv2026

Dual Inhibition of MYRF Cleavage by Its JM Region and PAN-1 CCT Gates Developmental Timing in C. elegans

Post-embryonic development proceeds through discrete stages, yet the mechanisms that coordinate transitions between stages remain incompletely defined. The transmembrane transcription factor MYRF undergoes intramolecular cleavage to release a nuclear-localized fragment essential for developmental progression in C. elegans. Previously, we showed that PAN-1 as a key partner required for MYRF activity during development. PAN-1 promotes MYRF trafficking to the cell membrane--an essential step for MYRF cleavage--through interactions between their extracellular domains (Xia et al., 2021). Here, we show that MYRF-1 cleavage and nuclear translocation oscillate with larval stage transitions in C. elegans, peaking mid-to-late stage and being suppressed during molts. Using endogenous gene editing and mutant reporters, we identify an uncharacterized juxtamembrane (JM) region in MYRF-1 that self-inhibits cleavage. JM deletion triggers premature MYRF-1 nuclear entry, early lin-4 activation, growth defects, and adult lethality. We further demonstrate that the cytoplasmic tail (CCT) of the transmembrane protein PAN-1 acts as a predominant trans-inhibitor of MYRF-1 cleavage, coupling extracellular association with cytoplasmic inhibition. PAN-1 CCT deletion causes near-constitutive MYRF-1 nuclear accumulation, leading to premature lin-4 activation, accelerated M-cell division, and larval lethality. Removing these inhibitory mechanisms on MYRF-1 cleavage overrides nutrient-responsive developmental checkpoints. These findings uncover dual inhibitory mechanisms governing MYRF-1 cleavage and establish MYRF-1 cleavage as a key gatekeeper of developmental timing.

developmental biology↗

bioRxiv2025

Improved identification of peptides, cross-link sites, and modification sites by Target-enhanced Accurate Inclusion Mass Screening (TAlMS)

Chemical cross-linking of proteins coupled with mass spectrometry provides structural insights by identifying cross-linked peptide pairs, abbreviated as cross-links. Presently, cross-link identification suffers from ambiguity and poor sensitivity, because they are typically of lower abundance and consequently of lower MS2 quality than linear peptides present in the same sample. Here, we present Target-enhanced Accurate Inclusion Mass Screening (TAIMS), a meticulously optimized targeted mass spectrometry method. TAIMS significantly improved the quality of MS2 as indicated by fragment ion coverage (FIC) and other metrics. From data-dependent acquisition (DDA) to TAIMS, high-FIC cross-links increased by 401 or 678 on yeast ribosome or E. coli lysate, respectively, or from about 40% to around 90%. As a result, TAIMS substantially enhanced the accuracy of cross-link site localization and mitigated sensitivity loss in cross-link identification from large database searches. Enhanced identification sensitivity of TAIMS is further evidenced by its capacity to recover genuine cross-link identifications from data that would typically be discarded. On cross-linked E. coli lysate, 10.5% (284/2711) of the inclusion-list entries generated from unidentified cross-link-spectrum matches gained identity through TAIMS. Of these, 230 were linear peptides and 54 were cross-links, including 10 inter-molecular cross-links missed entirely by DDA. Additionally, we demonstrate that TAIMS is a general method for identification of low-abundance, post-translationally modified peptides. On a mouse brain sample, TAIMS increased the number of phosphopeptides identified with accurate phosphosite assignment by 67%. These findings indicate that TAIMS holds broad applicability in proteomics.

biochemistry↗

bioRxiv2025

Nucleophagy is promoted by two autophagy receptors and inhibited by chromatin-nuclear envelope tethering in fission yeast

Selective autophagy of the nucleus, known as nucleophagy, targets nuclear components for degradation. The molecular mechanisms underlying nucleophagy remain inadequately understood. In this study, we identify a nucleophagy receptor, Npr1, in the fission yeast Schizosaccharomyces pombe. Npr1 is an Atg8-binding multi-transmembrane protein localized to the outer nuclear membrane. It functions redundantly with another autophagy receptor Epr1 to promote nitrogen starvation-induced nucleophagy. In the absence of both Npr1 and Epr1, starved cells exhibit abnormal nuclear morphology and reduced survival. During nucleophagy, the nuclear envelope (NE) forms outward protrusions where Atg8 co-localizes with Npr1 and/or Epr1. These protrusions subsequently detach from the NE, resulting in the formation of autophagosomes that contain nucleophagy cargo. Notably, artificially enhancing chromatin association with the inner nuclear membrane leads to NE protrusions that fail to detach, thereby aborting nucleophagy. Our findings provide mechanistic insights into nucleophagy and suggest that abortive nucleophagy protects chromatin from degradation.

cell biology↗

bioRxiv2024

Do-it-yourself de novo antibody sequencing workflow that achieves complete accuracy of the variable regions

Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable regions of an antibody--both the heavy chain and the light chain--is a prerequisite for the production of recombinant antibodies. Mass spectrometry-based de novo sequencing is a frequently used, and sometimes the only approach to gaining this information. Here, we describe a workflow that enables accurate sequence determination of monoclonal antibodies based on mass spectrometry data and freely available software tools. This workflow, which we developed using a homemade anti-FLAG monoclonal antibody as a reference sample, achieved 100% accuracy of the variable regions with clear distinction between leucine (L) and isoleucine (I). Using this workflow, we successfully decoded a monoclonal anti-HA antibody, for which we had no prior knowledge of its sequence. Based on the de novo sequencing result, we generated a recombinant anti-HA antibody, and demonstrated that it has the same specificity, sensitivity, and affinity as the commercial antibody. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/630060v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@180c502org.highwire.dtl.DTLVardef@1064399org.highwire.dtl.DTLVardef@4005d3org.highwire.dtl.DTLVardef@13e65ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Global analysis of protein and small-molecule substrates of ubiquitin-like proteins (UBLs)

AbstractUbiquitin-like proteins (UBLs) constitute a family of evolutionarily conserved proteins that share similarities with ubiquitin in 3D structures and modification mechanisms. For most UBLs including Small-Ubiquitin-like Modifiers (SUMO), their modification sites on substrate proteins cannot be identified using the mass spectrometry-based method that has been successful for identifying ubiquitination sites, unless a UBL protein is mutated accordingly. To identify UBL modification sites without having to mutate UBL, we have developed a dedicated search engine pLink-UBL on the basis of pLink, a software tool for identification of cross-linked peptide pairs. pLink-UBL exhibited superior precision, sensitivity, and speed than "make-do" search engines such as MaxQuant, pFind, and pLink. For example, compared to MaxQuant, pLink-UBL increased the number of identified SUMOylation sites by 50 [~] 300% from the same datasets. Additionally, we present a method for identifying small-molecule modifications of UBLs. This method involves antibody enrichment of a UBL C-terminal peptide following enrichment of a UBL protein, followed by LC-MS/MS analysis and a pFind 3 blind search to identify unexpected modifications. Using this method, we have discovered non-protein substrates of SUMO, of which spermidine is the major one for fission yeast SUMO Pmt3. Spermidine can be conjugated to the C-terminal carboxylate group of Pmt3 through its N1 or also likely, N8 amino group in the presence of SUMO E1, E2, and ATP. Pmt3-spermidine conjugation does not require E3 and can be reversed by SUMO isopeptidase Ulp1. SUMO-spermidine conjugation is present in mice and humans. Also, spermidine can be conjugated to ubiquitin in vitro by E1 and E2 in the presence of ATP. The above observations suggest that spermidine may be a common small molecule substrate of SUMO and possibly ubiquitin across eukaryotic species. HighlightsO_LIA specialized software pLink-UBL enables precise identification of UBL modification sites on protein substrates. C_LIO_LIpFind 3 blind search enables identification of unexpected small-molecule substrates of a UBL protein. C_LIO_LISpermidine is a small molecule substrate of fission yeast SUMO Pmt3 as well as mammalian SUMO proteins. C_LIO_LIThe C-terminal carboxyl group of Pmt3 can be attached to the N1 or likely also the N8 amino group of spermidine in the presence of E1, E2, and ATP, and can be detached by SUMO isopeptidase. C_LI

biochemistry↗

bioRxiv2024

A distant TANGO1 family member promotes vitellogenin export from the ER in C. elegans

Vitellogenin belongs to the Large Lipid Transfer Protein superfamily and is thought to share a common ancestor with human Apolipoprotein B (ApoB) for systemic lipid transport1-5. Vitellogenin forms part of yolk granules (also known as yolk organelles), as maternal contribution of nutrients that support the embryonic development of oviparous animals6,7. In Caenorhabditis elegans, vitellogenin proteins (VIT-1 to VIT-6) are synthesized in the hermaphrodite intestine, secreted into the pseudocoelom and internalized by oocytes8-13. Although a general route for inter-tissue vitellogenin transport has been described, the full mechanism that underlies its intracellular trafficking within the intestine remains obscure9,12,13. In humans, transport and Golgi organization protein 1 (TANGO1) and TANGO1-like (TALI) proteins generate super-sized membrane carriers to accommodate bulky ApoB-containing lipoprotein particles for their export from ER exit sites14-17. Transport facilitated by TANGO1 family of proteins is considered as an alternative, COPII-independent ER exit pathway18-24. Thus far, TANGO1 orthologs have been discovered in most metazoans, except nematodes24,25. Here, we report the C. elegans protein R148.3 (now Transport and Golgi organization 1-like or TNGL-1) as a mediator of vitellogenin export from the ER. TNGL-1 depletion triggers VIT-2 accumulation in the intestinal ER lumen. TNGL-1 requires its C-terminal unstructured domain for its localization to ER exit sites. Like TANGO1, it utilizes a luminal globular domain for cargo engagement. Our findings support TNGL-1 as a distant TANGO1 family member.

cell biology↗

bioRxiv2024

Multiple allostery in the regulation of PDGFR beta kinase activities

Platelet-derived growth factor receptor beta (PDGFR{beta}), a type III receptor tyrosine kinase (RTK) with a featured kinase insert, regulates important cellular functions. Dysregulation of PDGFR{beta} is associated with cardiovascular and fibrosis diseases. Thus, its kinase activity needs to be precisely regulated under physiological conditions. Early studies demonstrated that its kinase was autoinhibited by its juxtamembrane segment and activated by transphosphorylation. However, additional mechanisms are required for the comprehensive regulation of the receptor kinase. Herein, we provide evidences that dimerization of activated kinases, autoinhibition by the kinase insert, and dimerization of inactive kinase, all contribute to the regulation of the receptor kinase. Moreover, we find such multiple allosteric regulation is also conserved in other type III RTKs, including colony stimulating factor 1 receptor (CSF1R). Impairing the allosteric regulation of CSF1R is associated with malfunctions of microglia and demyelination of neurons in Hereditary Diffuse Leukoencephalopathy with Spheroids (HDLS).

biophysics↗

bioRxiv2024

Effect of the terminal phosphate group on negative-ion CID/HCD fragmentation behaviors of RNA oligonucleotides

An increasing need of mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigated the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either or both ends, or neither. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that depends on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5- or 3-phosphate, or both, have a higher charge state distribution and lose upon CID or HCD the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-). Such neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g. 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions), hence unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we have identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.

molecular biology↗

bioRxiv2023

Pan-sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of C. elegans

Multiple isozymes are encoded in the C. elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM), three important classes of sphingolipids from the same MS run. Validating this pan-sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3 plays a greater role than CGT-1 does in producing glucosylceramides. Intriguingly, lagr-1 RNAi lowers the abundance of all sphingomyelin species and that of several glucosylceramide species, which suggests that LAGR-1 may have functions beyond what is predicted. Additionally, RNAi of sms-1, -2, and -3 all lower the abundance of sphingomyelins with an odd number of carbon atoms (mostly C21 and C23, with or without hydroxylation) in the N-acyl chain, and only sms-1 RNAi does not elevate sphingomyelins containing even-numbered N-acyl chains. This suggests that sphingolipids containing even-numbered N-acyl chains could be regulated separately, sometimes in opposite directions, with those containing odd-numbered N-acyls, presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of glucosylceramides and sphingomyelins.

genetics↗

bioRxiv2023

ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and inhibits the Warburg effect in breast cancer

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked {beta}-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.

biochemistry↗

bioRxiv2023

Ubiquitination-mediated Golgi-to-endosome sorting determines the poison-antidote duality of wtf meiotic drivers

Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. Here we investigate how the toxin and antidote products of a fission yeast wtf-family KMD gene can act antagonistically. Both the toxin and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs N-terminal PY motifs (Leu/Pro-Pro-X-Tyr) to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby preventing it from causing toxicity. A physical interaction between the antidote and the toxin enables the ubiquitinated antidote to translocate the toxin to the endosome and neutralize its toxicity. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs.

cell biology↗

bioRxiv2023

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through a liquid-liquid phase separation. Here, we find that the phase separation of SYD-2/Liprin-, a key active zone scaffold, is controlled by phosphorylation. Using phosphoproteomics, we identify the SAD-1 kinase to phosphorylate SYD-2 and a number of other substrates. Presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. We determine SAD-1 phosphorylation of SYD-2 at three sites is critical to activate its phase separation. Mechanistically, phosphorylation relieves a binding interaction between two folded SYD-2 domains that inhibits phase separation by an intrinsically disordered region. We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, enabling its phase separation and active zone assembly.

neuroscience↗

bioRxiv2023

Signaling from the C. elegans Hypodermis Non-autonomously Facilitates Short-term Associative Memory

Memory loss is one of the most debilitating symptoms of aging. While we normally think of memory regulation as autonomous to the brain, other factors outside of the brain can also affect neuron function. We have shown that the longevity insulin/IGF-1 signaling (IIS) pathway regulates neuron-specific activity, but whether the maintenance of memory through the IIS pathway is completely autonomous to the nervous system, or if there are systemic inputs, is unknown. We address this question using the auxin-inducible degradation (AID) system to degrade the IIS receptor, DAF-2, in specific tissues. Surprisingly, DAF-2 degradation specifically in the hypodermis improves memory in both young and aged C. elegans through the hypodermal expression of the diffusible Notch ligand, OSM-11. The Notch receptor LIN-12 is required for OSM-11s effect on memory, as are the downstream transcription factor LAG-1 and co-activator SEL-8. Furthermore, mid-life overexpression of the Notch ligand OSM-11 improves memory and slows cognitive decline. Hypodermal DAF-2 degradation suppresses the expression of ins-19, whose downregulation extends memory. Together, our data suggest a model in which the hypodermis, a metabolic tissue, can non-autonomously regulate neuronal activity and function, indicating a systemic connection between metabolism and memory regulation.

genetics↗

bioRxiv2023

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and in epitranscriptomic studies. However, MS fragmentation behaviors of RNA oligos are understood insufficiently. In this study, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos of four to eight nucleotides (nt) in length by collision-induced dissociation (CID) using a high-resolution, accurate-mass instrument. We find that in the CID spectra acquired under the normalized collisional energy of 35%, [~]70% of the total peak intensity belonged to sequencing ions (a-B, a, b, c, d, w, x, y, z), [~]25% belonged to precursor ions with either complete or partial loss of a nucleobase in the form of a neutral or an anion, and the remainder were internal ions and anionic nucleobases. Of the sequencing ions, the most abundant species were y, c, w, a-B, and a ions. The charge state of the RNA precursor ions strongly affected their fragmentation behaviors. As the precursor charge increased from -1 to -5, the fractional intensity of sequencing ions in the CID spectra decreased, whereas the fractional intensity of precursor ions with neutral and/or charged losses of a nucleobase increased. Moreover, RNA oligos containing U, especially at the 3' terminus, tended to produce precursors that lost HNCO and/or NCO-, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings build a strong foundation for mechanistic understanding of RNA fragmentation by MS/MS, contributing to future automated identification of RNA oligos from their CID spectra in a more efficient way.

bioinformatics↗

bioRxiv2023

Comparative analysis of chemical cross-linking mass spectrometry data indicates that protein STY residues rarely react with N-hydroxysuccinimide ester cross-linkers

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) has enjoyed growing popularity in biomedical research. Most CXMS experiments utilize cross-linkers based on N-hydroxysuccinimide (NHS) ester, which react selectively with the amine groups found on the free N-termini of proteins and on the side chain of lysine (K) residues. It is also reported that under certain conditions they can react with the hydroxyl groups of serine (S), threonine (T), and tyrosine (Y). Some of the popular cross-link search engines including MeroX and xiSearch set STY, in addition to K, as cross-linkable sites by default. However, to what extent NHS ester cross-linkers react with STY under the typical CXMS experimental conditions remains unclear, nor has the reliability of STY-cross-link identifications. Here, by setting amino acids with chemically inert side chains such as glycine (G), valine (V), and leucine (L) as cross-linkable sites, which serves as a negative control, we show that software-identified STY-cross-links are only as reliable as GVL-cross-links. This is true across different NHS ester cross-linkers including DSS, DSSO, and DSBU, and across different search engines including MeroX, xiSearch, and pLink. Using a published dataset originated from synthetic peptides, we demonstrate that STY-cross-links indeed have a high false discovery rate. Further analysis revealed that depending on the data and the CXMS search engine used to analyze the data, up to 65% of the STY-cross-links identified are actually K-K cross-links of the same peptide pairs, up to 61% are actually K-mono-links, and the rest tend to contain short peptides at high risk of false identification.

biochemistry↗

bioRxiv2022

O-GlcNAcylation promotes YTHDF1 cytosolic localization and colorectal cancer tumorigenesis

O-linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalking with phosphorylation and ubiquitination to orchestrate various biological processes. Herein we show that it modifies the N6-methyladenosine (m6A)-mRNA reader YTHDF1 and fine-tunes its nuclear translocation by the exportin protein Crm1. First we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified the YTHDF1 O-GlcNAcylation sites to be Ser196/Ser197/Ser198, as described in previous numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196AS197FS198A (AFA) mutants, which significantly attentuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating the downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 might promote the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis might underlie colorectal cancer tumorigenesis.

biochemistry↗