This study shows that Parkinson’s disease–associated kinase LRRK2 is activated by inhibition of PIKfyve, which metabolizes phosphatidylinositol 3-phosphate, and reveals a novel mechanism of LRRK2 activation that requires interaction with phosphatidylinositol 3-phosphate, as well as CASM and Rab. LRRK2 is a Parkinson’s disease–associated kinase that phosphorylates Rab GTPases. Hyperactivation of LRRK2, which is thought to cause Parkinson’s disease, occurs on stressed endolysosomal membranes via conjugation of ATG8 to single membranes (CASM) and Rab, whereas the involvement of specific membrane lipids remained unclear. Here, we found that the inhibition of PIKfyve that metabolizes phosphatidylinositol 3-phosphate (PI3P) induces hyperactivation of LRRK2 and that additional inhibition of the class III phosphoinositide 3-kinase that generates PI3P suppressed LRRK2 hyperactivation. Cellular PI3P levels were indeed elevated when LRRK2 was activated. CASM-induced activation of LRRK2, but not CASM itself, was suppressed by phosphoinositide 3-kinase inhibitors, whereas inhibition of CASM or Rab12/Rab29 suppressed LRRK2 activation induced by PIKfyve inhibitors. LRRK2 and phospho-Rab10 were colocalized with PI3P on LAMP1-positive enlarged vacuoles, and LRRK2 directly bound PI3P in vitro via its N-terminal positively charged amino acids. Together, we propose an updated mechanism of LRRK2 hyperactivation that simultaneously requires CASM, Rab, and PI3P-rich membranes.
Unsupervised explainable AI identifies ∼2,000 oligonucleotide-usage zones in the human genome and predicts high-resolution chromosome banding from genome sequences, bridging genomics and cytogenetics. Unsupervised and explainable AI can uncover genomic features beyond human expectations. Oligonucleotides such as penta- and hexanucleotides often function as core binding motifs for regulatory proteins, and their usage provides powerful tools in functional genomics. We applied an unsupervised, explainable AI to their odds-ratio profiles of all 1-Mb euchromatic fragments in the human genome. Clustering using penta- or hexanucleotide odds ratios (1,024 or 4,096 variables) unexpectedly revealed nearly 2,000 distinct zones. CG-containing penta- and hexanucleotides (244 and 1,185 variables) produced comparable ∼2,000 zones, indicating fundamental genomic segmental structures. Human chromosomes exhibit Giemsa-banding patterns comprising 850 bands at prometaphase and ∼2,000 at prophase. Using single-nucleotide coordinates available for the 850 bands, we identified diagnostic oligonucleotides distinguishing Giemsa-negative and Giemsa-positive regions. Computational pseudo-band reconstruction based on these oligonucleotides generated genome segmentations that more closely paralleled the AI-derived ∼2,000 zones than the 850 bands. These findings indicate that AI captures characteristic sequence features of chromosomal bands and can predict high-resolution banding from genome sequences alone, thereby bridging classical cytogenetics and modern AI-based genomics.
This study examines the impacts of SNORD116 on the expression and RNA modifications of diacylglycerol kinase kappa , an enzyme implicated in neurogenesis and synaptic function that are likely relevant to aspects of the pathophysiology of Prader–Willi syndrome. SNORD116 is a small nucleolar RNA deficient in Prader–Willi syndrome individuals. The mechanisms by which SNORD116 conveys its effects are unknown. Here, transcriptomic analysis showed that diacylglycerol kinase kappa ( Dgkk ) expression was elevated in the hypothalamus of Snord116 -deficient mice. In silico analyses predicted a strong RNA:RNA interaction between Snord116 and Dgkk mRNA that is fully conserved in mouse and human. In vitro experiments in human cellular models demonstrated that SNORD116 mediates 2′-O-methylation of DGKK mRNA and regulates DGKK mRNA and protein expression. In Snord116 -deficient mouse neurons, increased Dgkk expression was associated with attenuated diacylglycerol-mediated activation of ERK and PKC, critical elements in neuronal signaling cascades. In mice, brain Dgkk expression peaks during late embryonic and early postnatal periods, coinciding with peak rates of neurogenesis; its expression in adults is restricted mainly to brain regions implicated in Prader–Willi syndrome pathophysiology, including the arcuate nucleus of the hypothalamus. Fasting acutely decreased hypothalamic Dgkk expression in wild-type mice. Increased Dgkk expression in Snord116 -deficient mice could reflect derangements in this pathway that affects both brain development and cell-specific functions.
Immobilized antigen induces an immune synapse, which is supported by Swiprosin-1/EFhd2 (EFhd2) in B and T cells. Here, we find that EFhd2 regulates mitochondrial fitness in the B cell synapse. Whereas activated EFhd2-deficient (KO) B cells show higher mitochondrial mass than wildtype (WT) B cells, this potential advantage reverses on coated anti-IgM antibodies: activated WT B cells enhance basal and maximal respiration as well as proton leak, with large increments of mitochondrial spare capacity, but EFhd2KO B cells fail to do so. Actin destabilization, but much more so microtubule destabilization, impairs mitochondrial up-regulation in WT, but not in EFhd2KO B cells. Conversely, microtubule stabilization, and to a lesser extent actin stabilization, restore mitochondrial spare capacity in EFhd2KO B cells at the BCR synapse. EFhd2KO B cells fail to coordinate the microtubule organization center, mitochondria and IgM BCRs upon synapse formation. Hence, microtubule-dependent mitochondrial organization at the immune synapse coordinates mitochondrial fitness in B cells via EFhd2.
The RNA-binding domain of the human GEMIN5 protein consists of an oligomerizing extended surface, including a serine-rich motif and a disordered acidic cluster that cooperatively regulate RNA binding. GEMIN5 is emerging as a multifunctional RNA-binding protein (RBP) linked to human disease. This RBP comprises a WD40 repeat domain at the N terminus. The C-terminal region (G5C) includes a tetratricopeptide repeat (TPR)-like dimerization module, a non-canonical RNA-binding site (RBS), and an intrinsically disordered region (IDR). Understanding the molecular basis for RNA binding is critical to decipher how GEMIN5 pathological variants impact on their functions. Here we show that the RNA-binding domain consists of an extended binding surface comprising a serine-rich loop and an acidic cluster within the IDR. The RNA binding ability measured in vitro and in soluble cellular extracts indicated that individually, ser-rich motif and acidic cluster mutants diminished RNA binding intensity. However, in the absence of the RBS region, the TPR-extended protein is sufficient to facilitate RNA binding. AlphaFold modeling of the TPR-extended protein shows the assembly of an elongated dimer bound to RNA. The same RBS is observed in the G5C–RNA oligomer model. We propose that the elongated dimer forms an extended binding surface, fine-tuning the RNA binding ability of the G5C architecture. Graphical Abstract lsa;10/1/e202603860/GA F1 GA
