Compartmentalized localization of perinuclear proteins within germ granules in C. elegans.

Germ granules, or nuage, are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei. C. elegans perinuclear germ granules are composed of multiple subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P -bodies, and E granules.

The Role of Polo-Like Kinase 1 (PLK1) O-GlcNAcylation in Mitosis.

Polo-like kinase 1 (PLK1) is a crucial mitotic kinase that is implicated in various aspects of cell cycle. Many post-translational modifications have been identified on PLK1 to regulate its activation, stability, and localization. PLK1 has been shown previously to colocalize with the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), and OGT regulates PLK1 stability.

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress.

O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus, and mitochondria. The recent advent of mass spectrometry technology has enabled the identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication, and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism.

A meiotic driver hijacks an epigenetic reader to disrupt mitosis in noncarrier offspring.

Killer meiotic drivers (KMDs) are selfish genetic elements that distort Mendelian inheritance by selectively killing meiotic products lacking the KMD element, thereby promoting their own propagation. Although KMDs have been found in diverse eukaryotes, only a limited number of them have been characterized at the molecular level, and their killing mechanisms remain largely unknown. In this study, we identify that a gene previously deemed essential for cell survival in the fission yeast is a single-gene KMD.

Cathepsin B promotes Aβ proteotoxicity by modulating aging regulating mechanisms.

While the activities of certain proteases promote proteostasis and prevent neurodegeneration-associated phenotypes, the protease cathepsin B (CTSB) enhances proteotoxicity in Alzheimer's disease (AD) model mice, and its levels are elevated in brains of AD patients. How CTSB exacerbates the toxicity of the AD-causing Amyloid β (Aβ) peptide is controversial. Using an activity-based probe, aging-altering interventions and the nematode C.

Effect of Terminal Phosphate Groups on Collisional Dissociation of RNA Oligonucleotide Anions.

The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate 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 end, both ends, or neither end.

Germ granule compartments coordinate specialized small RNA production.

Germ granules are biomolecular condensates present in most animal germ cells. One function of germ granules is to help maintain germ cell totipotency by organizing mRNA regulatory machinery, including small RNA-based gene regulatory pathways. The C.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes.

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans.

Multiple isozymes are encoded in the Caenorhabditis 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) from the same MS run. Validating this 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 both required for synthesis of the id17:1 sphingoid base.

ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates 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 β-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.

Test-Time Training for Deep MS/MS Spectrum Prediction Improves Peptide Identification.

In bottom-up proteomics, peptide-spectrum matching is critical for peptide and protein identification. Recently, deep learning models have been used to predict tandem mass spectra of peptides, enabling the calculation of similarity scores between the predicted and experimental spectra for peptide-spectrum matching. These models follow the supervised learning paradigm, which trains a general model using paired peptides and spectra from standard data sets and directly employs the model on experimental data.

Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of fission yeast 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. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation.

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 liquid-liquid phase separation. Here, we find that the phase separation of Caenorhabditis elegans SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation.

An EMC-Hpo-Yki axis maintains intestinal homeostasis under physiological and pathological conditions.

Balanced control of stem cell proliferation and differentiation underlines tissue homeostasis. Disruption of tissue homeostasis often results in many diseases. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological and pathological conditions remains poorly understood.

The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8.

Diamond controls epithelial polarity through the dynactin-dynein complex.

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells.

Comparative Analysis of Chemical Cross-Linking Mass Spectrometry Data Indicates That Protein STY Residues Rarely React with -Hydroxysuccinimide Ester Cross-Linkers.

When it comes to mass spectrometry data analysis for identification of peptide pairs linked by -hydroxysuccinimide (NHS) ester cross-linkers, search engines bifurcate in their setting of cross-linkable sites. Some restrict NHS ester cross-linkable sites to lysine (K) and protein N-terminus, referred to as K only for short, whereas others additionally include serine (S), threonine (T), and tyrosine (Y) by default. 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.

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 epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a high-resolution, accurate-mass instrument.

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-α, a key active zone scaffold, is controlled by phosphorylation.

Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes.

Transient and weak protein-protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers.

O-GlcNAcylation promotes the cytosolic localization of the mA reader YTHDF1 and colorectal cancer tumorigenesis.

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N-methyladenosine (mA) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the mA mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1.

Structural basis of membrane skeleton organization in red blood cells.

The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton.