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 DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

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. 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, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.

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. However, this approach can lead to inaccurate predictions due to differences between the training data and the experimental data, such as sample types, enzyme specificity, and instrument calibration. To tackle this problem, we developed a test-time training paradigm that adapts the pretrained model to generate experimental data-specific models, namely, PepT3. PepT3 yields a 10-40% increase in peptide identification depending on the variability in training and experimental data. Intriguingly, when applied to a patient-derived immunopeptidomic sample, PepT3 increases the identification of tumor-specific immunopeptide candidates by 60%. Two-thirds of the newly identified candidates are predicted to bind to the patient's human leukocyte antigen isoforms. To facilitate access of the model and all the results, we have archived all the intermediate files in Zenodo.org with identifier 8231084.

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. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent 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 the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a 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) and hence is 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 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.

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 ß-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. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. 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.

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. elegans germ granule is compartmentalized into multiple subcompartments whose biological functions are largely unknown. Here, we identify an uncharted subcompartment of the C. elegans germ granule, which we term the E granule. The E granule is nonrandomly positioned within the germ granule. We identify five proteins that localize to the E granule, including the RNA-dependent RNA polymerase (RdRP) EGO-1, the Dicer-related helicase DRH-3, the Tudor domain-containing protein EKL-1, and two intrinsically disordered proteins, EGC-1 and ELLI-1. Localization of EGO-1 to the E granule enables synthesis of a specialized class of 22G RNAs, which derive exclusively from 5' regions of a subset of germline-expressed mRNAs. Defects in E granule assembly elicit disordered production of endogenous siRNAs, which disturbs fertility and the RNAi response. Our results define a distinct subcompartment of the C. elegans germ granule and suggest that one function of germ granule compartmentalization is to facilitate the localized production of specialized classes of small regulatory RNAs.

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. Here we investigate how the toxin and antidote products of a 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 PY motifs (Leu/Pro-Pro-X-Tyr) in its N-terminal cytosolic tail 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.