SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.

SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence, and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.

Aquatic autotrophs that fix carbon using ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) frequently expend metabolic energy to pump inorganic carbon towards the enzyme’s active site. A central requirement of this strategy is the formation of highly concentrated Rubisco condensates known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes respectively. Recent data indicates these condensates form by the mechanism of liquid- liquid phase separation (LLPS). LLPS requires networks of weak multivalent interactions typically mediated by intrinsically disordered scaffold proteins. Here we comparatively review recent rapid developments that detail the determinants and precise interactions that underlie diverse Rubisco condensates. The burgeoning field of biomolecular condensates has few examples where LLPS can be linked to clear phenotypic outcomes. When present, Rubisco condensates are essential for photosynthesis and growth, and they are thus emerging as powerful and tractable models to investigate the structure function relationship of phase separation in biology.

Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco).A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism's method to increase substrate CO2 for Rubisco to achieve maximum functional capacity. Herein, we characterize part of the CCM of the diatom Phaeodactylum tricornutum through in vitro reconstitution of its pyrenoid. Studies prior to this project resulted in the discovery of an intrinsically disordered pyrenoid scaffold protein comprised of six tandem repeat sequences, Pyrenoid component 1 (PYCO1) which can form a dense phase to specifically recruit P. tricornutum Rubisco (PtRubisco). It was established that these interactions occur through "KWSPR/Q" motifs on PYCO1 with PtRubisco small subunits. In this study, more detailed characterization of these biomolecular condensates were carried out which elucidated several key findings. Rubisco was found to be enriched up to 200-fold in these condensates. Each PYCO1 molecule sequesters up to 3 units of Rubisco holoenzymes in fully-saturated condensates.The mobility of components in these condensates also change according to Rubisco occupancy. Cryo-EM structural analysis confirmed the interaction of the "KWSPR" motif with Rubisco. Additional motifs on the C-terminus of PYCO1 also interact with Rubisco through an alpha helical motif conserved within P.tricornutum. The data suggests that the "KWSPR" stickers must dimerize prior to binding the SSU of Rubisco. Specific aromatic residues on PYCO1 are required for both heterotypic and homotypic phase separation of the system.Additional pulldown experiments on pyrenoid components led to the identification of a novel protein with the accession number Phatr3_J47612.Phatr3_J47612 and its fragments partition into PYCO1 condensates and antagonize the recruitment of PtRubisco. The likely next steps in this project would be directed towards a systematic study of the interplay of several pyrenoidal proteins to develop our understanding of the diatom CCM.