Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4.

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function.

Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4.

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function.

The nuclear Argonaute HRDE-1 directs target gene re-localization and shuttles to nuage to promote small RNA-mediated inherited silencing.

Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei.

Case series of COVID-19 patients from the Qinghai-Tibetan Plateau Area in China.

Background: Coronavirus disease 2019 (COVID-19) is a serious infection caused by the new coronavirus severe acute respiratory syndrome coronavirus 2. The disease was first identified in December 2019 and has caused significant morbidity and mortality worldwide.

Aim: To explore the clinical characteristics and treatments for COVID-19 in the Qinghai-Tibetan Plateau Area in China.

PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in .

Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how germlines handle information have come from the study of factors that specify or maintain the germline fate. In early embryos, the CCCH zinc finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs.

HDAC1 SUMOylation promotes Argonaute-directed transcriptional silencing in .

Eukaryotic cells use guided search to coordinately control dispersed genetic elements. Argonaute proteins and their small RNA cofactors engage nascent RNAs and chromatin-associated proteins to direct transcriptional silencing. The small ubiquitin-like modifier (SUMO) has been shown to promote the formation and maintenance of silent chromatin (called heterochromatin) in yeast, plants, and animals.

CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase.

Improving mass spectrometry analysis of protein structures with arginine-selective chemical cross-linkers.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions.

A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art search engines.

Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine.

We present a sequence-tag-based search engine, Open-pFind, to identify peptides in an ultra-large search space that includes coeluting peptides, unexpected modifications and digestions. Our method detects peptides with higher precision and speed than seven other search engines. Open-pFind identified 70-85% of the tandem mass spectra in four large-scale datasets and 14,064 proteins, each supported by at least two protein-unique peptides, in a human proteome dataset.

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript.

Protocol for analyzing protein ensemble structures from chemical cross-links using DynaXL.

Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information.

Polη O-GlcNAcylation governs genome integrity during translesion DNA synthesis.

DNA polymerase η (Polη) facilitates translesion DNA synthesis (TLS) across ultraviolet (UV) irradiation- and cisplatin-induced DNA lesions implicated in skin carcinogenesis and chemoresistant phenotype formation, respectively. However, whether post-translational modifications of Polη are involved in these processes remains largely unknown. Here, we reported that human Polη undergoes O-GlcNAcylation at threonine 457 by O-GlcNAc transferase upon DNA damage.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous.

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation.

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole.

Phosphorylation of LSD1 by PLK1 promotes its chromatin release during mitosis.

Background: Lysine-specific histone demethylase 1 (LSD1) modulates chromatin status through demethylation of H3K4 and H3K9. It has been demonstrated that LSD1 is hyperphosphorylated and dissociates from chromatin during mitosis. However, the molecular mechanism of LSD1 detachment is unknown.

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones.

Structural characterization of coatomer in its cytosolic state.

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting.

Increasing the Depth of Mass-Spectrometry-Based Structural Analysis of Protein Complexes through the Use of Multiple Cross-Linkers.

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS(3) and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins.

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation.

ESCRTs Cooperate with a Selective Autophagy Receptor to Mediate Vacuolar Targeting of Soluble Cargos.

Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy.

Visualizing the Ensemble Structures of Protein Complexes Using Chemical Cross-Linking Coupled with Mass Spectrometry.

Abstract: Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-µmol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure.

Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24.

CAMKII and calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16.

The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C.