Stress Granules and FUS condensates recruit functionally distinct sets of RNAs

Cellular homeostasis relies on the organization of RNA and protein into membrane-less organelles. Stress granules (SGs) are well-known hubs for translational repression during cellular stress. Similar condensates formed by RNA-binding proteins such as FUS-- mutations in which cause amyotrophic lateral sclerosis (ALS)--remain poorly characterized relative to canonical SGs. Here, we develop Granule-seq, a microcapillary-based granule-resolved RNA sequencing approach, and demonstrate that SGs and FUS condensates are functionally distinct RNA compartments rather than variants of a unified granule class. Granules were individually aspirated, analyzed in small pooled sets, and validated through overlap with known SG components (794/2,759 genes, 28.8%, p < 0.001). Gene Ontology analysis revealed that G3BP-positive SGs sequester transcripts for stress responses and translational control, while FUS condensates are enriched for transcripts essential for synaptic function and neuronal development. Direct comparison showed that 63% of FUS-enriched and 82% of G3BP-enriched genes were granule-specific, with only 493 genes shared. Exploratory sequence analysis revealed modest contributions from compositional features, with functional identity providing primary selectivity. As a proof-of-concept study with limited biological replication, our results suggest that distinct granule identities are established through functionally specialized transcriptomes, a process that may be disrupted in ALS, and provide a framework for understanding RNA sorting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/726389v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b867dforg.highwire.dtl.DTLVardef@ab89d9org.highwire.dtl.DTLVardef@1e71c78org.highwire.dtl.DTLVardef@1fe23e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Granule-seq workflow and key findings.(Left) Individual cytoplasmic granules are isolated by microcapillary aspiration, pooled ([~]10 granules per replicate), and processed for RNA sequencing alongside total-cell input. (Middle) Venn diagram showing the overlap between FUS condensate-enriched (1,334 genes) and G3BP1 stress granule-enriched (2,759 genes) transcripts. FUS condensates preferentially recruit neuronal and synaptic transcripts; G3BP1 stress granules are enriched for stress-response and proteostasis transcripts. (Right) Two-step recruitment model: shared sequence features (long 3'UTR, GC-rich composition, stable RNA structure) provide permissiveness for granule entry (Step 1), while RBP interactome composition and functional context determine granule-type specificity (Step 2). C_FIG