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Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco's CO 2 fixation, yet hornwort Rubisco remains poorly characterized. Here we assembled the hornwort Anthoceros agrestis Rubisco (AaRubisco) using the Arabidopsis thaliana SynBio expression system and observed the formation of stalled intermediates, prompting us to develop a new SynBio system with A. agrestis cognate chaperones. We successfully assembled AaRubisco and Rubisco from three other hornwort species. Unlike A. thaliana Rubisco, AaRubisco assembly is not dependent on RbcX or Raf2. Kinetic characterization reveals that hornwort Rubiscos exhibit a range of catalytic rates (3-10 s -1 ), but with similar affinity (∼30 μM) and specificity (∼70) for CO 2 . These results suggest that hornwort Rubiscos do not comply with the long-held canonical catalytic trade-off observed in other land plants, providing experimental support that Rubisco kinetics may be phylogenetically constrained. Unexpectedly, we observed a 50% increase in AaRubisco catalytic rates when RbcX was removed from our SynBio system, without any reduction in specificity. Structural biology, biochemistry, and proteomic analysis suggest that subtle differences in Rubisco large-subunit interactions, when RbcX is absent during biogenesis, increases the accessibility of active sites and catalytic turnover rate. Collectively, this study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discoveries will contribute to developing new approaches for engineering Rubisco with superior kinetics., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Metagenomic analysis of the symbiotic cyanobacteria colonies within Gunnera tinctoria stems revealed a new strain of Nostoc . Here, we report its genome sequence., Competing Interests: The authors declare no conflict of interest.

The slow kinetics and poor substrate specificity of the key photosynthetic CO 2 -fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum . PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

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 (or Rubiscondensates) known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes, respectively. Recent data indicate that these condensates form by the mechanism of liquid-liquid phase separation. This mechanism 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 liquid-liquid phase separation 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)