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Hi-C chromatin maps of EZH2 WT and EZH2 Y646X lymphomas. EZH2 p.Tyr646* gain-of-function alterations lead to a genomewide increase in H3K27me3 (Supplementary Fig. 1a). To establish whether this global accumulation of H3K27me3 modifies the genome topology on a similarly broad scale, we performed high-throughput chromatin conformation capture (Hi-C) in two lymphoma cell lines (Karpas422 and WSU-DLCL2) expressing the mutant form of EZH2 (EZH2 Y646X) and a lymphoma cell line (OCI-Ly19) expressing the wild-type EZH2 (EZH2 WT) protein (Supplementary Table 1 and Supplementary Note). Contact maps of EZH2 Y646X and EZH2 WT cells were binned in regions of 50 kb and compared with multiple metrics (Fig. 1). For each pair of maps, we compared the overall distribution of chromosomal contacts by using the stratum-adjusted correlation coefficient (SCC) 24 (Fig. 1a); the fraction of 1-Mb regions assigned to the same compartment (A or B) 3 (Fig. 1b); the similarity among TADs 25-27 (Fig. 1c); and the fraction of bin interactions that were significant in both maps, also known as the cell interactome 28,29 (Fig. 1d). To build a reference scale of values for each metric, we compared Hi-C maps of EZH2 Y646X lymphoma cell lines with Hi-C maps of endothelial cells (HUVEC), fetal fibroblasts (IMR90), and normal lymphoblastoid cells (GM12878). Moreover, we used randomized contact maps or

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesised RNA, RNA processing proteins, and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs) 1 – 3 . Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localisation microscopy and correlative electron microscopy, and find that MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates 4 . Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

authors contributed equally 10 15 20 2 Mitochondria contain the genetic information and expression machinery to produce proteins essential for cellular respiration. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins, and mitoribosome assembly factors are known to form punctate subcompartments referred to as mitochondrial RNA granules (MRGs) 1-3 . Despite their proposed role in regulating gene expression, little is known about the structural 5 and dynamic properties of MRGs. We investigated the organization of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy techniques, obtaining ultrastructural details of their internal architecture. We find that MRGs are organized into nanoscale RNA cores surrounded by a protein shell. Using livecell super-resolution structured illumination microscopy and photobleaching perturbations, 10 we reveal that MRGs undergo fusion and rapidly exchange components, consistent with liquid-liquid phase separation (LLPS). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with membrane remodeling. Inhibition of mitochondrial fission leads to an aberrant distribution of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. 15 Together, our results reveal a role for LLPS in concentrating RNA and its processing proteins into MRGs, which are positioned along mitochondria by membrane dynamics.RNA in eukaryotic and bacterial cells is sequestered into granules that exhibit a wide range of forms and functions, under both physiological and stress conditions. For example, nuclear speckles and paraspeckles, are involved in RNA splicing and transcriptional regulation 4-6 . The 20 nucleolus creates a compartment for ribosomal assembly 7,8 , and in the cytoplasm, stress granules protect mRNAs during cellular stress 9 . RNA-protein granules often form by liquid-liquid phase separation 10 . In developing embryos, the sensitivity of P granule formation by LLPS to the local 3 concentration of proteins leads to symmetry breaking and differential inheritance of material between germ and somatic cells 11 . Multivalent weak interactions between disordered RNAbinding protein (RBP) domains, and RNA itself, were identified as crucial factors for the formation of biomolecular condensates in many in vitro and in silico studies 12-15 . To understand their biological function, in vivo studies are essential; phase behavior is sensitive to elements of the 5 molecular environment such as salt concentration, pH, or crowding, and physiological conditions are challenging to reproduce in test tubes 16 .MRGs are ribonucleoprotein complexes composed of newly synthesized long polycistronic mtRNAs, originating from the 16kb mitochondrial DNA (mtDNA), together with several mitochondrial RBPs 1,17,18 . It was previously demonstrated that mtRNA is essential for MRG 10 formation 3 . However, both the structural organization of and the dynamic interplay betwee...