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To explore the mechanism of Rab5/RAB-5 activation during endocytic recycling, we perform a genome-wide RNAi screen and identify a recycling regulator, LET-502/ROCK. LET-502 preferentially interacts with RAB-5(GDP) and activates RABX-5 GEF activity toward RAB-5, presumably by disrupting the self-inhibiting conformation of RABX-5. Furthermore, we find that the concomitant loss of LET-502 and another CED-10 effector, TBC-2/RAB-5-GAP, results in an endosomal buildup of RAB-5, indicating that CED-10 directs TBC-2-mediated RAB-5 inactivation and re-activates RAB-5 via LET-502 afterward. Then, we compare the functional position of LET-502 with that of RME-6/RAB-5-GEF. Loss of LET-502-RABX-5 module or RME-6 leads to diminished RAB-5 presence in spatially distinct endosome groups. We conclude that in the intestine of C. elegans, RAB-5 resides in discrete endosome subpopulations. Under the oversight of CED-10, LET-502 synergizes with RABX-5 to revitalize RAB-5 on a subset of endosomes in the deep cytosol, ensuring the progress of basolateral recycling.Oncoproteins such as the BRAFV600E kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAFV600E-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAFV600E in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAFV600E-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAFV600E-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAFV600E.ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. [https://www.selleckchem.com/products/arn-509.html find more] Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to &gt;5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.Although cell transplantation can rescue motor defects in Parkinson's disease (PD) models, whether and how grafts functionally repair damaged neural circuitry in the adult brain is not known. We transplanted hESC-derived midbrain dopamine (mDA) or cortical glutamate neurons into the substantia nigra or striatum of a mouse PD model and found extensive graft integration with host circuitry. Axonal pathfinding toward the dorsal striatum was determined by the identity of the grafted neurons, and anatomical presynaptic inputs were largely dependent on graft location, whereas inhibitory versus excitatory input was dictated by the identity of grafted neurons. hESC-derived mDA neurons display A9 characteristics and restore functionality of the reconstructed nigrostriatal circuit to mediate improvements in motor function. These results indicate similarity in cell-type-specific pre- and post-synaptic integration between transplant-reconstructed circuit and endogenous neural networks, highlighting the capacity of hPSC-derived neuron subtypes for specific circuit repair and functional restoration in the adult brain.