Regulation of spatially restricted gene expression: linking RNA localization and phase separation.

Subcellular restriction of gene expression is crucial to the functioning of a wide variety of cell types. The cellular machinery driving spatially restricted gene expression has been studied for many years, but recent advances have highlighted novel mechanisms by which cells can generate subcellular microenvironments with specialized gene expression profiles. Particularly intriguing are recent findings that phase separation plays a role in certain RNA localization pathways. The burgeoning field of phase separation has revolutionized how we view cellular compartmentalization, revealing that, in addition to membrane-bound organelles, phase-separated cytoplasmic microenvironments - termed biomolecular condensates - are compositionally and functionally distinct from the surrounding cytoplasm, without the need for a lipid membrane. The coupling of phase separation and RNA localization allows for precise subcellular targeting, robust translational repression and dynamic recruitment of accessory proteins. Despite the growing interest in the intersection between RNA localization and phase separation, it remains to be seen how exactly components of the localization machinery, particularly motor proteins, are able to associate with these biomolecular condensates. Further studies of the formation, function, and transport of biomolecular condensates promise to provide a new mechanistic understanding of how cells restrict gene expression at a subcellular level.

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the additive effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

L-bodies are RNA-protein condensates driving RNA localization in Xenopus oocytes.

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

CRISPR/Cas9 Mutagenesis Reveals Versatile Roles of Hox Genes in Crustacean Limb Specification and Evolution.

Crustaceans possess a diverse array of specialized limbs. Although shifts in Hox gene expression domains have been postulated to play a role in generating this limb diversity, little functional data have been provided to understand the precise roles of Hox genes during crustacean development. We used a combination of CRISPR/Cas9-targeted mutagenesis and RNAi knockdown to decipher the function of the six Hox genes expressed in the developing mouth and trunk of the amphipod Parhyale hawaiensis. These experimentally manipulated animals display specific and striking homeotic transformations. We found that abdominal-A (abd-A) and Abdominal-B (Abd-B) are required for proper posterior patterning, with knockout of Abd-B resulting in an animal with thoracic type legs along what would have been an abdomen, and abd-A disruption generating a simplified body plan characterized by a loss of specialization in both abdominal and thoracic appendages. In the thorax, Ubx is necessary for gill development and for repression of gnathal fate, and Antp dictates claw morphology. In the mouth, Scr and Antp confer the part-gnathal, part-thoracic hybrid identity of the maxilliped, and Scr and Dfd prevent antennal identity in posterior head segments. Our results allow us to define the role Hox genes play in specifying each appendage type in Parhyale, including the modular nature by which some appendages are patterned by Hox gene inputs. In addition, we define how changes in Hox gene expression have generated morphological differences between crustacean species. Finally, we also highlight the utility of CRISPR/Cas9-based somatic mutagenesis in emerging model organisms.